CN119301253A - New Staple Nucleic Acid - Google Patents

Abstract

The subject of the present invention is to develop a Staple nucleic acid that can form a guanine quadruple chain structure with a target nucleic acid even when there are no 4 guanine repeat sequences on the target nucleic acid. The subject of the present invention is also to develop various uses of existing Staple nucleic acids and the Staple nucleic acids of the present invention. The inventors of the present invention solved the above-mentioned problem by developing the following oligonucleotides (second-generation Staple nucleic acids), which use oligonucleotides to supply guanine repeat sequences, resulting in a guanine quadruple chain structure being able to be formed on the target nucleic acid by the guanine repeats on the target nucleic acid and the guanine repeat sequences on the oligonucleotide totaling 4 guanine repeat sequences. The present invention also develops new uses for Staple nucleic acids for first-generation Staple nucleic acids and second-generation Staple nucleic acids.

Description

New Staple Nucleic Acid

Technical Field

The present invention relates to the development of new staple nucleic acids and the use of conventional staple nucleic acids and new staple nucleic acids.

Background Art

As gene expression inhibition technology, various tools such as microRNA and siRNA are known (non-patent document 1, non-patent document 2). If the sequence information of the target gene is known, siRNA can be designed relatively easily, and multiple target sequences can be selected for one target mRNA by referring to existing reports on the target gene, thereby strongly inhibiting gene expression.

For any technology, since it exerts its gene expression inhibition function through a simple mechanism of binding to mRNA, unexpected effects (off-target effects) are inevitable and become a problem (Non-Patent Document 3), which covers issues that must be resolved in order to be used in the human body.

With the improvement of current bioinformatics technology, the expression inhibition of off-target genes (off-target effect) has been able to be reduced to a minimum. However, from the perspective of pharmaceuticalization, the development of siRNA has not been smooth. The reason is that nucleic acid drugs need to use modified nucleic acids. It is known that when using modified nucleic acids, the microRNA effect and siRNA activity will be significantly reduced, and the problem of not being able to obtain the desired protein translation inhibition effect will also arise (non-patent literature 4).

In order to solve these problems, various artificial modified nucleic acids have been synthesized, but the current situation is that the development of artificial modified nucleic acids with high versatility as drugs is still ongoing. The biggest reason why artificial modified nucleic acids are difficult to develop is that siRNA needs to be linked to an enzyme reaction with target mRNA cleavage activity, that is, by chemically modifying nucleic acids to obtain nuclease resistance, on the other hand, the ability to be recognized as an enzyme reaction substrate is lost.

In order to solve these problems, the inventors of the present invention have previously developed a staple nucleic acid that has the function of binding to a sequence-specific target mRNA sequence and inducing the formation of a guanine quadruple chain structure using the four guanine repeat sequences present on the target mRNA, and developed a method for inhibiting the peptide synthesis reaction of the ribosome and inhibiting the protein translation reaction by utilizing the guanine quadruple chain structure induced by the staple nucleic acid (Patent Document 1).

Conventional staple nucleic acids require four guanine repeat sequences on the target mRNA and can only form a guanine quartet structure for target mRNAs with such a structure.

Prior art literature

Patent Literature

Patent document 1: WO2020/085510

Non-patent literature

Non-patent document 1: Fire, A., et al., Nature 1998, 391, 806.

Non-patent document 2: Ambros, V. Nature 2004, 431, 350.

Non-patent document 3: Jackson, A.L. and Linsley, P.S. Nature reviews. Drug discovery 2010, 9, 57.

Non-patent document 4: Jackson, A.L., et al., RNA 2006, 12, 1197.

Summary of the invention

Problem that the invention aims to solve

The present invention aims to develop a staple nucleic acid that can form a guanine quartet structure for a target nucleic acid even when there are no four guanine repeats in the target nucleic acid. The present invention also aims to develop various uses of existing staple nucleic acids and the staple nucleic acid of the present invention.

Solutions for solving problems

The inventors of the present invention solved the above-mentioned problems by developing a Staple nucleic acid (second-generation Staple nucleic acid), wherein the Staple nucleic acid supplies a guanine repeat sequence through an oligonucleotide, resulting in the formation of a guanine quadruple structure on the target nucleic acid using a total of four guanine repeat sequences, namely, the guanine repeat sequence on the target nucleic acid and the guanine repeat sequence on the oligonucleotide. The present invention also developed new uses of Staple nucleic acids for the first-generation Staple nucleic acids and the second-generation Staple nucleic acids.

More specifically, this application provides the following means to solve the above-mentioned problems:

[1]: A G-donating oligonucleotide comprising: a first nucleotide sequence and a second nucleotide sequence that hybridize to a nucleotide sequence on the 5′ side or the 3′ side of a nucleotide sequence containing 1 to 3 guanine repeat sequences on a target nucleic acid; and 3 to 1 guanine repeat sequence depending on the number of guanine repeat sequences on the target nucleic acid,

The G-donating oligonucleotide can change the three-dimensional structure of the target nucleic acid by hybridizing the first nucleotide sequence and the second nucleotide sequence with the target nucleic acid, so that the spatial distance between the guanine repeat sequence on the target nucleic acid and the guanine repeat sequence on the G-donating oligonucleotide, which is a total of four guanine repeat sequences, is shortened, and a guanine quadruple chain structure is formed through these four guanine repeat sequences;

[2]: The G-donating oligonucleotide according to [1], wherein the first nucleotide sequence and the second nucleotide sequence of the G-donating oligonucleotide are in a sequence that is identical to the guanine repeat sequence on the target nucleic acid.

· The nucleotide sequence portion on the 5' side or 3' side of the guanine repeat sequence on the 3' terminal side,

· The nucleotide sequence portion on the 5' or 3' side of the guanine repeat sequence on the 5' terminal side

to carry out hybridization;

[3]: The G-donating oligonucleotide according to [1] or [2], which is DNA, RNA, a modified nucleic acid, or a combination thereof;

[4]: A pharmaceutical composition comprising a G-donating oligonucleotide, wherein the G-donating oligonucleotide comprises: a first nucleotide sequence and a second nucleotide sequence that hybridize with a nucleotide sequence on the 5' side or the 3' side of the guanine repeat sequence, with respect to a nucleotide sequence portion comprising 1 to 3 guanine repeat sequences on a target nucleic acid; and 3 to 1 guanine repeat sequence depending on the number of guanine repeat sequences on the target nucleic acid,

The G-donating oligonucleotide can change the three-dimensional structure of the target nucleic acid by hybridizing the first nucleotide sequence and the second nucleotide sequence with the target nucleic acid, so that the spatial distance between the guanine repeat sequence on the target nucleic acid and the guanine repeat sequence on the G-donating oligonucleotide, which is a total of four guanine repeat sequences, is shortened, and a guanine quadruple chain structure is formed through these four guanine repeat sequences;

[5]: The pharmaceutical composition according to [4], wherein the first nucleotide sequence and the second nucleotide sequence of the G-donating oligonucleotide are in a manner similar to the guanine repeat sequence on the target nucleic acid.

· The nucleotide sequence portion on the 5' side or 3' side of the guanine repeat sequence on the 3' terminal side,

· The nucleotide sequence portion on the 5' or 3' side of the guanine repeat sequence on the 5' terminal side

to carry out hybridization;

[6]: The pharmaceutical composition according to [4] or [5], wherein the nucleic acid is DNA, RNA, a modified nucleic acid or a combination thereof;

[7]: A method for producing a G-donating oligonucleotide, characterized in that the G-donating oligonucleotide comprises: a first nucleotide sequence and a second nucleotide sequence that hybridize with a nucleotide sequence on the 5' side or the 3' side of the guanine repeat sequence, with respect to a nucleotide sequence portion containing 1 to 3 guanine repeat sequences on a target nucleic acid; and 3 to 1 guanine repeat sequence depending on the number of guanine repeat sequences on the target nucleic acid,

The method designs and synthesizes a G-donating oligonucleotide as follows: the G-donating oligonucleotide is folded in such a manner that the first nucleotide sequence and the second nucleotide sequence of the G-donating oligonucleotide hybridize with the target nucleic acid, thereby shortening the spatial distance between the guanine repeat sequences on the target nucleic acid and the guanine repeat sequences on the G-donating oligonucleotide, which are a total of four guanine repeat sequences;

[8]: The method for producing a G-donating oligonucleotide according to [7], wherein the first nucleotide sequence and the second nucleotide sequence of the G-donating oligonucleotide are in a sequence that is identical to the guanine repeat sequence on the target nucleic acid.

· The nucleotide sequence portion on the 5' side or 3' side of the guanine repeat sequence on the 3' terminal side,

· The nucleotide sequence portion on the 5' or 3' side of the guanine repeat sequence on the 5' terminal side

to carry out hybridization;

[9]: The method for producing a G-donating oligonucleotide according to [7] or [8], wherein the oligonucleotide is DNA, RNA, a modified nucleic acid, or a combination thereof;

[10]: A method for inhibiting protein expression from a target nucleic acid, wherein a G-donating oligonucleotide changes the three-dimensional structure of the target nucleic acid by hybridizing the first nucleotide sequence and the second nucleotide sequence with the target nucleic acid, thereby shortening the spatial distance between the guanine repeat sequences on the target nucleic acid and the guanine repeat sequences on the G-donating oligonucleotide, which together form a guanine quadruple chain structure, thereby inhibiting protein expression from the target nucleic acid.

The G-donating oligonucleotide comprises: a first nucleotide sequence and a second nucleotide sequence that hybridize with a nucleotide sequence on the 5' side or 3' side of the guanine repeat sequence for a nucleotide sequence portion comprising 1 to 3 guanine repeat sequences on the target nucleic acid; and 3 to 1 guanine repeat sequence depending on the number of guanine repeat sequences on the target nucleic acid;

[11]: The method for inhibiting protein expression according to [11], wherein the first nucleotide sequence and the second nucleotide sequence of the G-donating oligonucleotide are mutually exclusive with the guanine repeat sequence on the target nucleic acid.

· The nucleotide sequence portion on the 5' side or 3' side of the guanine repeat sequence on the 3' terminal side,

· The nucleotide sequence portion on the 5' or 3' side of the guanine repeat sequence on the 5' terminal side

to carry out hybridization;

[12]: The method for inhibiting protein expression according to [10] or [11], wherein the nucleic acid is DNA, RNA, a modified nucleic acid or a combination thereof;

[13]: The method for inhibiting protein expression according to [10] or [11], wherein the inhibition of protein expression is based on inhibition of a reverse transcription reaction or inhibition of a protein translation reaction.

[14]: A protein expression inhibition kit comprising a G-donating oligonucleotide,

The G-donating oligonucleotide comprises: a first nucleotide sequence and a second nucleotide sequence that hybridize with a nucleotide sequence on the 5' side or the 3' side of the guanine repeat sequence for a nucleotide sequence portion comprising 1 to 3 guanine repeat sequences on the target nucleic acid; and 3 to 1 guanine repeat sequence depending on the number of guanine repeat sequences on the target nucleic acid,

The G-donating oligonucleotide can change the three-dimensional structure of the target nucleic acid by hybridizing the first nucleotide sequence and the second nucleotide sequence with the target nucleic acid, so that the spatial distance between the guanine repeat sequence on the target nucleic acid and the guanine repeat sequence on the G-donating oligonucleotide, which is a total of four guanine repeat sequences, is shortened, and these four guanine repeat sequences form a guanine quadruple chain structure, thereby inhibiting the expression of protein by the target nucleic acid;

[15]: The protein expression inhibition kit according to [14], wherein the first nucleotide sequence and the second nucleotide sequence of the G-donating oligonucleotide are in a manner similar to the guanine repeat sequence on the target nucleic acid.

· The nucleotide sequence portion on the 5' side or 3' side of the guanine repeat sequence on the 3' terminal side,

· The nucleotide sequence portion on the 5' or 3' side of the guanine repeat sequence on the 5' terminal side

to carry out hybridization;

[16]: The protein expression inhibition kit according to [14] or [15], wherein the inhibition of protein expression is based on the inhibition of a reverse transcription reaction or the inhibition of a protein translation reaction.

[17]: A method for producing a modified protein from a target nucleic acid, wherein an oligonucleotide changes the three-dimensional structure of a target nucleic acid by hybridizing a first nucleotide sequence and a second nucleotide sequence with the target nucleic acid, thereby shortening the spatial distance between a total of four guanine repeat sequences on the target nucleic acid and the guanine repeat sequences on the oligonucleotide, and the four guanine repeat sequences form a guanine quartet structure, thereby causing a portion of the target nucleic acid to form a loop portion, shunting ribosomes to the loop portion, and producing a modified protein from the target nucleic acid.

The oligonucleotide comprises: a first nucleotide sequence and a second nucleotide sequence that hybridize with a nucleotide sequence on the 5' side or the 3' side of the guanine repeat sequence for a nucleotide sequence portion comprising 1 to 4 guanine repeat sequences on the target nucleic acid; and 3 to 0 guanine repeat sequences depending on the number of guanine repeat sequences on the target nucleic acid;

[18]: The method for producing a modified protein according to [17], wherein the first nucleotide sequence and the second nucleotide sequence of the oligonucleotide are homologous to the guanine repeat sequence on the target nucleic acid.

· The nucleotide sequence portion on the 5' side or 3' side of the guanine repeat sequence on the 3' terminal side,

· The nucleotide sequence portion on the 5' or 3' side of the guanine repeat sequence on the 5' terminal side

to carry out hybridization;

[19]: The method for producing a modified protein according to [17] or [18], wherein the nucleic acid is DNA, RNA, a modified nucleic acid or a combination thereof;

[20]: A method for producing a fusion protein, wherein the first nucleotide sequence in an oligonucleotide hybridizes with a first target nucleic acid, the second nucleotide sequence hybridizes with a second target nucleic acid, and the oligonucleotide changes the three-dimensional structure of these target nucleic acids, so that the spatial distance between 1 to 2 guanine repeat sequences on the first target nucleic acid, 1 to 2 guanine repeat sequences on the second target nucleic acid, and 2 to 0 guanine repeat sequences on the oligonucleotide is shortened, and these 4 guanine repeat sequences form a guanine quartet structure, thereby shunting ribosomes between the first target nucleic acid and the second target nucleic acid, thereby producing a fusion protein from a translation product of a partial sequence of the first target nucleic acid and a translation product of a partial sequence of the second target nucleic acid,

The oligonucleotide comprises:

A first nucleotide sequence that hybridizes with a nucleotide sequence on the 5' side or 3' side of the guanine repeat sequence for a nucleotide sequence portion containing one to two guanine repeat sequences on the first target nucleic acid,

A second nucleotide sequence that hybridizes with a nucleotide sequence on the 5' side or 3' side of the guanine repeat sequence to a nucleotide sequence portion containing one to two guanine repeat sequences on the second target nucleic acid, and

2 to 0 guanine repeat sequences depending on the number of guanine repeat sequences on the first target nucleic acid and the number of guanine repeat sequences on the second target nucleic acid;

[21]: The method for producing a fusion protein according to [20], wherein the first nucleotide sequence and the second nucleotide sequence of the oligonucleotide are homologous to the guanine repeat sequence on the target nucleic acid.

· The nucleotide sequence portion on the 5' side or 3' side of the guanine repeat sequence on the 3' terminal side,

· The nucleotide sequence portion on the 5' or 3' side of the guanine repeat sequence on the 5' terminal side

to carry out hybridization;

[22]: The method for producing a fusion protein according to [20] or [21], wherein the nucleic acid is DNA, RNA, a modified nucleic acid or a combination thereof;

[23]: A method for stabilizing a target nucleic acid, wherein an oligonucleotide changes the three-dimensional structure of the target nucleic acid by hybridizing the first nucleotide sequence and the second nucleotide sequence with the target nucleic acid, thereby shortening the spatial distance between the guanine repeat sequences on the target nucleic acid and the guanine repeat sequences on the oligonucleotide, and the four guanine repeat sequences form a guanine quadruple chain structure, thereby inhibiting the binding of a nuclease to the target nucleic acid or inhibiting the function of a nuclease, thereby stabilizing the target nucleic acid.

The oligonucleotide comprises: a first nucleotide sequence and a second nucleotide sequence that hybridize with a nucleotide sequence on the 5' side or the 3' side of the guanine repeat sequence for a nucleotide sequence portion comprising 1 to 4 guanine repeat sequences on the target nucleic acid; and 3 to 0 guanine repeat sequences depending on the number of guanine repeat sequences on the target nucleic acid;

[24]: The method for stabilizing a target nucleic acid according to [23], wherein the first nucleotide sequence and the second nucleotide sequence of the oligonucleotide are mutually exclusive with the guanine repeat sequence on the target nucleic acid.

· The nucleotide sequence portion on the 5' side or 3' side of the guanine repeat sequence on the 3' terminal side,

· The nucleotide sequence portion on the 5' or 3' side of the guanine repeat sequence on the 5' terminal side

to carry out hybridization;

[25]: The method for stabilizing a target nucleic acid according to [29] or [30], wherein the target nucleic acid is DNA, RNA, a modified nucleic acid, or a combination thereof.

Effects of the Invention

The present invention can develop and provide a new oligonucleotide (second-generation Staple nucleic acid), which supplies guanine repeat sequences through oligonucleotides, resulting in the formation of a guanine quadruple chain structure on the target nucleic acid through a total of 4 guanine repeat sequences on the target nucleic acid and the guanine repeat sequences on the oligonucleotide. The present invention can also provide new uses of oligonucleotides for first-generation Staple nucleic acids and second-generation Staple nucleic acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the formation of a guanine quartet structure when the binding mode of a G-donating staple nucleic acid is changed.

Fig. 2 is a diagram showing the construction of a guanine quartet structure using G-donating staple nucleic acids in the model sequences 2+1 100nt, 1+1 100nt_mut 1, and 1+1 100nt_mut 2. Here, (A) shows the results of fluorescence measurement using Thioflavin T for m2+1100nt RNA, (B) shows the results of fluorescence measurement using Thioflavin T for 1+1100nt_mut 1 RNA, and (C) shows the results of fluorescence measurement using Thioflavin T for 1+1100nt_mut 2 RNA.

Fig. 3 is a diagram showing the determination of the formation position of the guanine quartet structure in various target nucleic acid sequences. Here, (A) shows the results of the stop test of the 1+1 100nt RNA sequence, and (B) shows the results of the stop test of the TRPC65'UTR RNA sequence.

Fig. 4 is a diagram showing the results of studying the length of the G-supplying staple nucleic acid and the T-linker in the TRPC6 5'UTR sequence. Here, (A) shows the results of the stop test when the number of T-linkers in the G-supplying staple nucleic acid is changed, and (B) shows the results of the stop test when the length of the G-supplying staple nucleic acid is changed.

Fig. 5 is a diagram showing the evaluation results of the effect of the guanine quartet structure constructed using a G-donating staple nucleic acid on the protein translation reaction. Here, (A) shows a schematic diagram of each nucleic acid template used in in vitro translation, and (B) shows the in vitro translation results of the TRPC6 5'UTR sequence (n=2).

FIG. 6 is a graph showing the evaluation results of whether the presence or absence of a staple nucleic acid improves the stability of a target nucleic acid.

7 is a graph showing the results of comparing the GFP signal intensities of PTC GFP (no staple nucleic acid), Δtype1 GFP (no staple nucleic acid), PTC GFP (Staple nucleic acid 1), and PTC GFP (Staple nucleic acid 2), with the GFP signal intensity when GFP mRNA was translated being set to 100.

FIG8-1 is a graph showing that the stability of a target nucleic acid is significantly improved due to the presence of a Staple nucleic acid.

FIG. 8-2 is a graph showing that, when cells are used, the stability of the target nucleic acid is improved due to the presence of the staple nucleic acid, resulting in an increase in protein expression in the cells.

FIG. 9-1 is a graph showing that the stability of a target nucleic acid is significantly improved due to the presence of a Staple nucleic acid.

FIG. 9-2 is a graph showing that, when cells are used, the stability of the target nucleic acid is improved due to the presence of the staple nucleic acid, resulting in an increase in protein expression in the cells.

FIG. 10 is a diagram showing that the Staple nucleic acid of the present invention can cause ribosome shunting within a single molecule.

FIG. 11 is a diagram showing that the Staple nucleic acid of the present invention can cause ribosome shunt between two molecules.

DETAILED DESCRIPTION

The terms used in the present invention are defined as follows:

(a) Staple nucleic acid: refers to an oligonucleotide having the following functions: hybridizing with two sequences on a sequence-specific target nucleic acid to change the three-dimensional structure of the target nucleic acid, and inducing the formation of a guanine quadruple chain structure using the guanine repeat sequence present on the target nucleic acid. There are non-G-donating Staple nucleic acids (non-G-donating oligonucleotides, also called first-generation Staple nucleic acids) and G-donating Staple nucleic acids (G-donating oligonucleotides, also called second-generation Staple nucleic acids) described later;

(b) Non-G-donating Staple nucleic acid: It is a type of Staple nucleic acid, which refers to a Staple nucleic acid produced with reference to the Staple nucleic acid disclosed in Patent Document 1 and does not contain a guanine repeat sequence in the Staple nucleic acid itself. It refers to an oligonucleotide having the following functions: hybridizing with two sequences on a sequence-specific target nucleic acid to change the three-dimensional structure of the target nucleic acid, shortening the spatial distance of the four guanine repeat sequences on the target nucleic acid to induce the formation of a guanine quadruple chain structure. The non-G-donating Staple nucleic acid in the present invention is also called a non-G-donating oligonucleotide or a first-generation Staple nucleic acid.

(c) G-donating Staple nucleic acid: A type of Staple nucleic acid, which refers to a Staple nucleic acid newly disclosed in the present invention, which contains at least one guanine repeat sequence in the Staple nucleic acid itself. It refers to an oligonucleotide having the following functions: hybridizing with two sequences on a sequence-specific target nucleic acid to change the three-dimensional structure of the target nucleic acid, shortening the spatial distance between the guanine repeat sequence existing on the target nucleic acid and the guanine repeat sequence existing in the G-donating Staple nucleic acid, a total of four guanine repeat sequences, to induce the formation of a guanine quadruple chain structure. The G-donating Staple nucleic acid in the present invention is also called a G-donating oligonucleotide or a second-generation Staple nucleic acid.

(d) Nucleic acid: The nucleic acid constituting the staple nucleic acid in the present invention refers to any of naturally occurring nucleic acids or non-naturally occurring nucleic acids (non-natural nucleic acids). Naturally occurring nucleic acids refer to DNA or RNA composed of nucleic acid bases (i.e., adenine (a), guanine (g), cytosine (c), thymine (t), and uracil (u)). Non-naturally occurring nucleic acids (non-natural nucleic acids) refer to nucleic acids whose physical properties are changed by modifying the nucleic acid base, sugar, and phosphodiester moieties, and are also called modified nucleic acids.

The structure of the first generation of Staple nucleic acid

The first generation Staple nucleic acid in the present invention refers to a Staple nucleic acid prepared by referring to the Staple nucleic acid disclosed in Patent Document 1. That is, the first generation Staple nucleic acid has a structural feature that the Staple nucleic acid itself does not contain a guanine repeat sequence constituting a guanine quartet structure.

The first generation Staple nucleic acid in the present invention refers to: comprising: a first nucleotide sequence and a second nucleotide sequence that hybridize with a nucleotide sequence near (i.e., on the 5' side or the 3' side) any two guanine repeat sequences in the guanine repeat sequence to a nucleotide sequence portion containing four guanine repeat sequences on the target nucleic acid, and

An oligonucleotide having the function of changing the three-dimensional structure of the target nucleic acid hybridized with the first nucleotide sequence and the second nucleotide sequence, shortening the spatial distance between four guanine repeating sequences on the target nucleic acid, and forming a guanine quadruple chain structure through the four guanine repeating sequences.

Generally, the guanine quartet structure in cells refers to a stable nucleic acid higher-order structure constructed when guanine repeat sequences of three or more bases are present in four locations in a spatial arrangement close to each other with less than seven bases. The inventors of the present invention have found that even if guanine repeat sequences are present at positions separated by an interval of seven or more bases on a target nucleic acid, using an appropriately designed oligonucleotide of the present invention comprising the first nucleotide sequence and the second nucleotide sequence, it is possible to artificially bring the guanine repeat sequences present in these target nucleic acids into close proximity in space, thereby inducing an artificial guanine quartet structure in the target nucleic acid.

In the present invention, when referring to the object to which the first generation Staple nucleic acid binds, that is, the guanine repeat sequence in the target nucleic acid, it refers to a continuous sequence containing more than 3 guanine bases present on the target nucleic acid, or a sequence containing more than 3 guanine bases in a nucleotide sequence that forms a protruding structure between guanines. Specific guanine repeat sequences include, for example, GGG, GNGG (N can be any base, and the number can be 1 or more), etc.

In the present invention, when referring to a target nucleic acid for which the first generation staple nucleic acid forms a guanine quartet structure, the target nucleic acid may be either RNA or DNA. The target nucleic acid is determined according to the intended use of the staple nucleic acid.

For the first generation Staple nucleic acid of the present invention to form a guanine quadruple chain structure in cells, the oligonucleotide needs to have:

The ability to recognize the sequence of the target nucleic acid, and

Ability to induce the formation of guanine quartet structures.

The ability of the first-generation Staple nucleic acid to recognize the target nucleic acid sequence can be achieved by comprising a first nucleotide sequence and a second nucleotide sequence that hybridize with a nucleotide sequence near the guanine repeat sequence on the target nucleic acid (i.e., on the 5' side or the 3' side) as the oligonucleotide of the first-generation Staple nucleic acid.

In the present invention, the sequences of the first nucleotide sequence and the second nucleotide sequence in the first-generation Staple nucleic acid are different for each target nucleic acid sequence. Two sequence portions on the target nucleic acid can be selected in a manner that can shorten the spatial distance between the four guanine repeat sequences on the target nucleic acid, and the first nucleotide sequence and the second nucleotide sequence can be determined in a manner that can hybridize with these two sequence portions.

The first nucleotide sequence and the second nucleotide sequence of the first-generation Staple nucleic acid can be appropriately designed. For example, without limitation, as an example of the first-generation Staple nucleic acid of the present invention, a nucleotide sequence on the downstream side (3' side) of the guanine repeat sequence of the target nucleic acid (for example, a region of 13 to 20 bases starting from a base within 20 bases immediately downstream (3' side) (i.e., the first base to the 20th base on the downstream side of the guanine repeat sequence)) or a nucleotide sequence starting from a base within 20 bases upstream (5' side) of the guanine repeat sequence of the target nucleic acid (i.e., the first base to the 20th base on the downstream side of the guanine repeat sequence) (for example, a region of 13 to 20 bases upstream (5' side)) can be selected, and the first nucleotide sequence and the second nucleotide sequence can be designed in a manner that can hybridize with these regions.

The distance between the nucleotide sequence region on the target nucleic acid to which the first nucleotide sequence and the second nucleotide sequence hybridize and the guanine repeat sequence of the target nucleic acid (the number of nucleotides between the nearest nucleotide of the first nucleotide sequence or the second nucleotide sequence when viewed from the guanine repeat sequence and the G of the guanine repeat sequence) can be appropriately determined by thermodynamic stability-related calculations, and the distance (number of nucleotides) is not particularly limited. According to the research of the present inventors, the formation of a guanine quadruple chain structure was confirmed even at a distance of 20 nucleotides. Therefore, as the starting point of the region to which the Staple nucleic acid hybridizes, it is possible to select: preferably, bases within 20 bases from the immediately downstream side (3' side) or the immediately upstream side (5' side) of the guanine repeat sequence (i.e., the 1st base to the 20th base from the downstream side or the upstream side of the guanine repeat sequence), more preferably, bases within 15 bases from the immediately downstream side (3' side) or the immediately upstream side (5' side) of the guanine repeat sequence (i.e., the 1st base to the 15th base from the downstream side or the upstream side of the guanine repeat sequence), more preferably, bases within 15 bases from the immediately downstream side (3' side) or the immediately upstream side (5' side) of the guanine repeat sequence. Bases within 10 bases from the immediate upstream side (5' side) (i.e., the 1st to 10th bases on the downstream or upstream side of the guanine repeat sequence), more preferably bases within 5 bases from the immediate downstream side (3' side) or the immediate upstream side (5' side) of the guanine repeat sequence (i.e., the 1st to 5th bases on the downstream or upstream side of the guanine repeat sequence), more preferably bases within 3 bases from the immediate downstream side (3' side) or the immediate upstream side (5' side) of the guanine repeat sequence (i.e., the 1st to 3rd bases on the downstream or upstream side of the guanine repeat sequence).

The first generation Staple nucleic acid can be composed of DNA, RNA, modified nucleic acid or a combination thereof. In the first generation Staple nucleic acid of the present invention, the first nucleotide sequence and the second nucleotide sequence are directly bound or indirectly bound via a linker. In the present invention, a preferred nucleic acid form can be selected for the first nucleotide sequence and the second nucleotide sequence, and the respective regions of the linker that can be included as needed.

Here, in the first generation Staple nucleic acid, when referring to the linker connecting the first nucleotide sequence and the second nucleotide sequence, it refers to a linker of a nucleic acid having any number of bases that exists between the first nucleotide sequence and the second nucleotide sequence. On the basis of the binding of the first nucleotide sequence to the target nucleic acid and the binding of the second nucleotide sequence to the target nucleic acid, the first generation Staple nucleic acid needs to have a length that can change the three-dimensional structure of the target nucleic acid, thereby shortening the spatial distance of the four guanine repeating sequences on the target nucleic acid.

The first generation of Staple nucleic acid is formed by directly combining the first nucleotide sequence and the second nucleotide sequence or indirectly combining them with a linker. The overall length of the Staple nucleic acid varies according to the nucleotide sequence of the target nucleic acid. It can be any length designed based on the GC content, Tm value and other benchmarks generally known in the technical field. According to its design, a longer nucleotide length can also be implemented. As an example, the overall length of the Staple nucleic acid can be 26 to 40 nucleotides in length, but is not limited to this length. The length of the region where the Staple nucleic acid hybridizes to the target nucleic acid can be the same or different from the length of the above-mentioned one end side (e.g., 3' side) region and the other end side (e.g., 5' side) region, without limitation.

The hybridization reaction between the first generation Staple nucleic acid and the target nucleic acid designed as described above can be carried out in any conditions in vivo or in vitro, for example:

The Staple nucleic acid is mixed into a solution containing the target nucleic acid and annealed;

This is done by simply mixing the Staple nucleic acid into a solution containing the target nucleic acid;

· This is carried out by introducing a short hairpin expression vector and expressing the staple nucleic acid from within the cell.

The hybridization reaction between the target nucleic acid and the staple nucleic acid can be carried out at a reaction temperature of 24 to 55° C. for, for example, 1 minute to 2 days.

In addition, the ability of the first-generation Staple nucleic acid to induce the formation of a guanine quadruple chain structure can be achieved as follows: there are four guanine repeat sequences on the target nucleic acid, and the Staple nucleic acid hybridized to the target nucleic acid changes the three-dimensional structure of the target nucleic acid, thereby shortening the spatial distance between the four guanine repeat sequences on the above-mentioned target nucleic acid, resulting in inducing the four guanine repeat sequences on the target nucleic acid to form a guanine quadruple chain structure.

The guanine quartet structure formation reaction using the first generation Staple nucleic acid of the present invention can be carried out, for example, by causing a hybridization reaction between the target nucleic acid and the Staple nucleic acid and leaving it to stand in this state. The temperature and time of the guanine quartet structure formation reaction can be the same as the temperature and time of the hybridization reaction between the target nucleic acid and the Staple nucleic acid, for example, it can be 1 minute to 2 days at a reaction temperature of 24 to 55° C. For example, the formation of the guanine quartet structure can be confirmed by an in vitro translation test, a fluorescent signal determination method based on a reaction with thioflavin T, a stop test method, etc.

An example of the specific structure of the first generation staple nucleic acid is shown in the following Chemical 1. The oligonucleotide described as "short-chain nucleic acid" in Chemical 1 refers to a staple nucleic acid, and the introduction of this oligonucleotide brings four guanine repeat sequences located on the target nucleic acid closer together, inducing the formation of a guanine quartet structure.

[Chemistry 1]

The structure of the second generation Staple nucleic acid

The second generation Staple nucleic acid in the present invention refers to a Staple nucleic acid in which a part of the four guanine repeat sequences constituting the guanine quartet structure is provided by an oligonucleotide. That is, the second generation Staple nucleic acid specifically has a structural feature that the Staple nucleic acid itself contains a guanine repeat sequence.

The second generation staple nucleic acid in the present invention refers to: a G-donating oligonucleotide, comprising: a first nucleotide sequence and a second nucleotide sequence that hybridize with a nucleotide sequence near (i.e., on the 5' side or the 3' side) any two guanine repeat sequences in the guanine repeat sequence, with respect to a nucleotide sequence portion comprising 1 to 3 guanine repeat sequences on the target nucleic acid; and 3 to 1 guanine repeat sequence depending on the number of guanine repeat sequences on the target nucleic acid,

The G-donating oligonucleotide can change the three-dimensional structure of the target nucleic acid by hybridizing the first nucleotide sequence and the second nucleotide sequence with the target nucleic acid, thereby shortening the spatial distance between the guanine repeat sequences on the target nucleic acid and the guanine repeat sequences on the G-donating oligonucleotide, which total four guanine repeat sequences, and forming a guanine quadruple chain structure through these four guanine repeat sequences.

Here, when referring to "containing 1 to 3 guanine repeating sequences on the target nucleic acid", it means that at least 1 guanine repeating sequence for forming a guanine quartet structure is present on the target nucleic acid, and 4 or more guanine repeating sequences may be present on the target nucleic acid. When there are 4 or more guanine repeating sequences on the target nucleic acid, it means that the guanine quartet structure is formed by 1 to 3 guanine repeating sequences among these guanine repeating sequences. In addition, "the number of guanine repeating sequences on the target nucleic acid" refers to the number of guanine repeating sequences on the target nucleic acid used to form a guanine quartet structure using the second-generation Staple nucleic acid of the present invention. For example, even if there are 4 or more guanine repeating sequences on the target nucleic acid, when a guanine quartet structure is formed using 2 of the guanine repeating sequences and 2 guanine repeating sequences supplied by a G supplying oligonucleotide, the "number of guanine repeating sequences on the target nucleic acid" is 2.

As described above, generally, the guanine quartet structure in cells is a stable nucleic acid higher-order structure constructed when guanine repeat sequences of three or more bases are present at four locations in a spatial arrangement close to each other with less than seven bases. The inventors of the present invention have found that, when there are 1 to 3 guanine repeat sequences present at positions separated by an interval of 7 or more bases on the target nucleic acid, by using a G-donating oligonucleotide comprising the appropriately designed first nucleotide sequence and second nucleotide sequence of the present invention and 3 to 1 guanine repeat sequence depending on the number of guanine repeat sequences on the target nucleic acid, the 1 to 3 guanine repeat sequences present in the target nucleic acid and the 3 to 1 guanine repeat sequence present in the G-donating oligonucleotide are artificially placed close to each other in space, thereby inducing an artificial guanine quartet structure in the target nucleic acid.

In the present invention, when referring to the object to which the second-generation Staple nucleic acid binds, that is, the guanine repeat sequence in the target nucleic acid, it refers to a continuous sequence of 3 or more guanine bases present on the target nucleic acid or a continuous sequence of 3 or more guanine bases present on the Staple nucleic acid. Specific guanine repeat sequences include guanine repeat sequences that can serve as elements of the formation of a guanine quadruple chain structure, such as GGG, GNGG (N represents an arbitrary nucleotide), etc., as listed in the paper reports.

In the present invention, as in the case of the first generation Staple nucleic acid, when referring to the target nucleic acid for the second generation Staple nucleic acid to form a guanine quartet structure, the target nucleic acid may be either RNA or DNA. The nucleic acid type of the target nucleic acid is determined according to the intended use of the Staple nucleic acid.

For the second generation Staple nucleic acid of the present invention to form a guanine quadruple chain structure in cells, the oligonucleotide needs to have:

Ability to recognize the sequence of target nucleic acid;

The ability to supply guanine repeat sequences so that the total number of guanine repeat sequences is 4, depending on the number of guanine repeat sequences present in the target nucleic acid (wherein, when there are 4 or more guanine repeat sequences in the target nucleic acid, 1 to 3 guanine repeat sequences among these guanine repeat sequences are used); and

Ability to induce the formation of guanine quartet structures.

The ability of the second-generation Staple nucleic acid to recognize the sequence of the target nucleic acid can be achieved by comprising a first nucleotide sequence and a second nucleotide sequence as an oligonucleotide of the second-generation Staple nucleic acid that hybridizes with a nucleotide sequence near the guanine repeat sequence on the target nucleic acid (i.e., on the 5' side or the 3' side).

In the present invention, the sequences of the first nucleotide sequence and the second nucleotide sequence in the second-generation staple nucleic acid are different for each target nucleic acid sequence. Two sequence portions on the target nucleic acid can be selected in a manner that can shorten the spatial distance between 1 to 3 guanine repeat sequences on the target nucleic acid and 3 to 1 guanine repeat sequences on the second-generation staple nucleic acid, a total of four guanine repeat sequences, and the first nucleotide sequence and the second nucleotide sequence can be determined in a manner that can hybridize with these two sequence portions.

The first nucleotide sequence and the second nucleotide sequence of the second generation Staple nucleic acid can be appropriately designed. For example, the first nucleotide sequence and the second nucleotide sequence of the Staple nucleic acid can be selected to be identical to the guanine repeat sequence on the target nucleic acid,

The nucleotide sequence portion near the guanine repeat sequence closest to the 3' end (i.e., the 5' side or the 3' side),

The nucleotide sequence portion near the guanine repeat sequence closest to the 5' end (i.e., the 5' side or the 3' side)

Sequences to be hybridized.

For example, as an example of the second-generation Staple nucleic acid of the present invention, a region of 13 to 20 bases on the immediate downstream side (3' side) of the guanine repeat sequence of the target nucleic acid and a region of 13 to 20 bases on the upstream side (5' side) of the guanine repeat sequence of the target nucleic acid are selected, and the first nucleotide sequence and the second nucleotide sequence are designed in a manner to hybridize with these regions.

The first nucleotide sequence and the second nucleotide sequence of the second generation Staple nucleic acid of the present invention can be appropriately designed according to its use. For example, it can be appropriately designed to have the following characteristics (but not limited to these):

At least one of the first nucleotide sequence or the second nucleotide sequence hybridizes with a nucleotide sequence portion near a guanine repeat sequence present before a stop codon on the target nucleic acid;

At least one of the first nucleotide sequence or the second nucleotide sequence hybridizes with a nucleotide sequence portion near a guanine repeat sequence present in the 5' untranslated region of the target nucleic acid;

The first nucleotide sequence and the second nucleotide sequence both hybridize to a nucleotide sequence portion near the guanine repeat sequence present in the 5' untranslated region of the target nucleic acid;

The first nucleotide sequence and the second nucleotide sequence both hybridize to a portion of the nucleotide sequence near the guanine repeat sequence present in the 3' untranslated region,

When used for other purposes, it can be appropriately designed according to the purpose.

The distance between the nucleotide sequence region on the target nucleic acid to which the first nucleotide sequence and the second nucleotide sequence hybridize and the guanine repeat sequence of the target nucleic acid (the number of nucleotides between the nearest nucleotide of the first nucleotide sequence or the second nucleotide sequence when viewed from the guanine repeat sequence and the G of the guanine repeat sequence) can be appropriately determined by thermodynamic stability-related calculations, and the distance (number of nucleotides) is not particularly limited. According to the research of the present inventors, the formation of a guanine quadruple chain structure was confirmed even at a distance of 20 nucleotides.

The second generation Staple nucleic acid can be composed of DNA, RNA, modified nucleic acid or a combination thereof. The second generation Staple nucleic acid of the present invention is formed by directly combining the first nucleotide sequence and the second nucleotide sequence and 3 to 1 guanine repeat sequences or indirectly combining them with a linker. In the present invention, a preferred nucleic acid form can be selected for each region of the first nucleotide sequence and the second nucleotide sequence, 3 to 1 guanine repeat sequences and the linker that can be included as needed.

Here, in the case of the second generation Staple nucleic acid, when referring to the linker connecting the first nucleotide sequence and the second nucleotide sequence, it refers to a linker of a nucleic acid with any base number other than the first nucleotide sequence, the second nucleotide sequence, and the guanine repeat sequence, which exists between the first nucleotide sequence and/or the second nucleotide sequence and the guanine repeat sequence on the Staple nucleic acid, and between the guanine repeat sequence on the Staple nucleic acid. On the basis of the binding of the first nucleotide sequence to the target nucleic acid and the binding of the second nucleotide sequence to the target nucleic acid, the second generation Staple nucleic acid needs to have a length that can change the three-dimensional structure of the target nucleic acid, thereby shortening the spatial distance between the guanine repeat sequence on the target nucleic acid and the guanine repeat sequence on the Staple nucleic acid, which is a total of four guanine repeat sequences.

The second generation Staple nucleic acid is formed by directly combining the first nucleotide sequence and the second nucleotide sequence and the guanine repeat sequence at 3 to 1 or indirectly combining them with a linker. The overall length of the Staple nucleic acid varies according to the nucleotide sequence of the target nucleic acid. For example, it can be any length designed based on the GC content, Tm value and other benchmarks generally known in the technical field. As an example, the overall length of the Staple nucleic acid can be 26 to 40 nucleotides in length, but is not limited to these lengths. The length of the region where the Staple nucleic acid hybridizes to the target nucleic acid can be the same or different from the length of the above-mentioned one end side (e.g., 3' side) region and the other end side (e.g., 5' side) region, without limitation.

The hybridization reaction between the second generation staple nucleic acid designed as described above and the target nucleic acid can be carried out in vivo or in vitro in the same manner as the first generation staple nucleic acid, for example:

The Staple nucleic acid is mixed into a solution containing the target nucleic acid and annealed;

This is done by simply mixing the Staple nucleic acid into a solution containing the target nucleic acid;

· This is carried out by introducing a short hairpin expression vector and expressing the Staple nucleic acid from within the cell.

The hybridization reaction between the target nucleic acid and the staple nucleic acid can be carried out at a reaction temperature of 24 to 55° C. for, for example, 1 minute to 2 days.

In one embodiment of the present invention, the second-generation staple nucleic acid is characterized in that it has the ability to supply guanine repeat sequences in a manner that the total number of guanine repeat sequences is 4 according to the number of guanine repeat sequences present in the target nucleic acid. That is, regardless of the number of guanine repeat sequences present in the target nucleic acid, when a guanine quartet structure is formed using any one guanine repeat sequence present in the target nucleic acid, the staple nucleic acid can supply guanine repeat sequences at three locations, when a guanine quartet structure is formed using any two guanine repeat sequences present in the target nucleic acid, the staple nucleic acid can supply guanine repeat sequences at two locations, and when a guanine quartet structure is formed using any three guanine repeat sequences present in the target nucleic acid, the staple nucleic acid can supply guanine repeat sequences at one location.

Therefore, since the staple nucleic acid supplies the guanine repeat sequence for forming the guanine quartet structure, the second-generation staple nucleic acid is an oligonucleotide that supplies the guanine repeat sequence, and in this sense can also be called a G-supplying oligonucleotide or a G-supplying staple nucleic acid.

In addition, the ability of the second-generation Staple nucleic acid to form a guanine quadruple chain structure can be achieved as follows: there are 4 guanine repeat sequences in total, namely, the guanine repeat sequences in the target nucleic acid and the guanine repeat sequences in the second-generation Staple nucleic acid; the Staple nucleic acid hybridized to the target nucleic acid changes the three-dimensional structure of the target nucleic acid, thereby shortening the spatial distance between the 4 guanine repeat sequences in total on the target nucleic acid and the second-generation Staple nucleic acid, thereby inducing the formation of a guanine quadruple chain structure among the 4 guanine repeat sequences in total on the target nucleic acid and the second-generation Staple nucleic acid.

The guanine quartet structure formation reaction using the second generation Staple nucleic acid of the present invention can be carried out, for example, by causing a hybridization reaction between the target nucleic acid and the Staple nucleic acid and leaving it to stand in this state. The temperature and time of the guanine quartet structure formation reaction can be the same as the temperature and time of the hybridization reaction between the target nucleic acid and the Staple nucleic acid, for example, it can be a reaction temperature of 24 to 55° C., for example, 1 minute to 2 days. For example, the formation of the guanine quartet structure can be confirmed by an in vitro translation test, a fluorescent signal determination method based on a reaction with thioflavin T, a stop test method, etc.

An example of a specific structure is shown in the following Chemical Formula 2. In this Chemical Formula 2, pattern A shows the case where the oligonucleotide supplies three guanine repeat sequences, pattern B shows the case where the oligonucleotide supplies two guanine repeat sequences, and pattern C shows the case where the oligonucleotide supplies one guanine repeat sequence. By introducing the oligonucleotide, the spatial distance of the guanine repeat sequences on the target nucleic acid and the guanine repeat sequences on the G supplying oligonucleotide, a total of four guanine repeat sequences, is shortened, and the four guanine repeat sequences are induced to form a guanine quadruple chain structure.

【Chemistry 2】

Method for producing the second generation Staple nucleic acid

The present invention can also provide a method for producing the second-generation staple nucleic acid (G-donating oligonucleotide).

Specifically, a method for producing a G-donating oligonucleotide can be provided, characterized in that the G-donating oligonucleotide comprises: a first nucleotide sequence and a second nucleotide sequence that hybridize with a nucleotide sequence near (i.e., on the 5' side or the 3' side) the guanine repeat sequence for a nucleotide sequence portion comprising 1 to 3 guanine repeat sequences on a target nucleic acid; and 3 to 1 guanine repeat sequence depending on the number of guanine repeat sequences on the target nucleic acid,

The method designs and synthesizes a G-donating oligonucleotide as follows: the G-donating oligonucleotide is folded in such a way that the first nucleotide sequence and the second nucleotide sequence of the G-donating oligonucleotide hybridize with the target nucleic acid, thereby shortening the spatial distance between the guanine repeat sequences on the target nucleic acid and the guanine repeat sequences on the G-donating oligonucleotide, which total four guanine repeat sequences.

In the manufacture of the second generation Staple nucleic acid of the present invention, it is important to appropriately design the structure of the Staple nucleic acid, that is, it is important to appropriately design the first nucleotide sequence and the second nucleotide sequence for hybridizing with the target nucleic acid, the number of guanine repeat sequences provided, and the linker that connects these sequence parts that may be included according to the situation. When designing the sequence constituting the Staple nucleic acid, it is preferably designed in a manner that reflects the characteristics of the second generation Staple nucleic acid described above. If the structure of the Staple nucleic acid can be designed, the Staple nucleic acid itself can be synthesized using a conventional nucleic acid synthesis method.

Functions of oligonucleotides

As a result of forming a guanine quartet structure, the first-generation Staple nucleic acid and the second-generation Staple nucleic acid of the present invention can both exert the same function. That is, the function exerted by the first-generation Staple nucleic acid can also be exerted by the second-generation Staple nucleic acid, and the function exerted by the second-generation Staple nucleic acid can also be exerted by the first-generation Staple nucleic acid. This is because the function of the Staple nucleic acid of the present invention is generated as a result of forming a guanine quartet structure, and the properties of the guanine quartet structure formed by either the first-generation Staple nucleic acid or the second-generation Staple nucleic acid are the same.

(1) Protein expression inhibition

In the present invention, as a result of forming a guanine quartet structure on the target nucleic acid, the expression of protein from the target nucleic acid can be inhibited.

As a specific example of a method for inhibiting protein expression, for example, as a result of forming a guanine quartet structure, the ribosome cannot bind to the target nucleic acid (mRNA) or the ribosome bound to the target nucleic acid (mRNA) stops moving in the 5'→3' direction at the position of the guanine quartet structure, thereby inhibiting the protein translation reaction, resulting in the inhibition of protein expression. In order to exert such a function, for example, the sequence of the Staple nucleic acid of the present invention can be designed in such a way that a guanine quartet structure is formed in the 5'UTR region and the open reading frame region on the target nucleic acid to exert the above function.

As shown in Patent Document 1 as an example of the first generation of Staple nucleic acid, the oligonucleotide having this structure can inhibit the protein translation reaction by forming a guanine quartet structure formed by the participation of the guanine repeat sequence present in the 5' untranslated region (5'UTR) of the target nucleic acid, and as a result, it is manifested as an enzyme-independent protein translation inhibition system (Chemical 3, upper left figure). This is because, even if the ribosome binds to the 5'UTR of the target nucleic acid, the ribosome cannot move to the 3' side of the target nucleic acid due to the guanine quartet structure, thereby stopping protein translation.

In addition, as a specific example of other methods for inhibiting protein expression, the formation of a guanine quartet structure results in the inability of reverse transcriptase to bind to the target nucleic acid (RNA) or the reverse transcriptase bound to the target nucleic acid (RNA) stops moving in the 3'→5' direction at the position of the guanine quartet structure, resulting in the inhibition of the protein expression reaction.

Therefore, the present invention can provide:

A method for inhibiting protein expression from a target nucleic acid, wherein a G-donating oligonucleotide changes the three-dimensional structure of the target nucleic acid by hybridizing a first nucleotide sequence and a second nucleotide sequence with the target nucleic acid, shortening the spatial distance between a total of four guanine repeat sequences on the target nucleic acid and the guanine repeat sequence on the G-donating oligonucleotide, and the four guanine repeat sequences form a guanine quadruple chain structure, thereby inhibiting protein expression from the target nucleic acid.

The G-donating oligonucleotide comprises: a nucleotide sequence portion containing 1 to 3 guanine repeat sequences on the target nucleic acid, a first nucleotide sequence and a second nucleotide sequence that hybridize with a nucleotide sequence near (i.e., on the 5' side or 3' side) the guanine repeat sequence; and 3 to 1 guanine repeat sequence depending on the number of guanine repeat sequences on the target nucleic acid.

The present invention discloses that: when a guanine quartet structure is formed by second-generation oligonucleotides and the guanine quartet structure formed by these oligonucleotides is formed by a guanine repeat sequence present in the 5' untranslated region (5'UTR) of the target nucleic acid, the inhibitory function of the protein expression shown here is also produced.

(2) Stabilization of target nucleic acid

In another example shown in Chemical 3, the following situation is shown: the result of forming a guanine quartet structure is that, for example, the movement of a nucleic acid hydrolase such as an exonuclease bound to mRNA in the 5' direction will stop at the position of the guanine quartet structure, and the target nucleic acid decomposition reaction is inhibited. This is because, when the guanine quartet structure is formed by 4 guanine repeat sequences including the guanine repeat sequence present in the 3' untranslated region (3'UTR) of the target nucleic acid, the exonuclease cannot move to the 5' side of the target nucleic acid due to the guanine quartet structure, inhibiting the target nucleic acid decomposition effect of the 3'→5' exonuclease (Chemical 3, upper right figure). In order to exert such a function, for example, when the target nucleic acid is mRNA, the sequence of the Staple nucleic acid of the present invention can be designed in a manner that forms a guanine quartet structure in the 3'UTR region on the target nucleic acid, thereby exerting the above-mentioned function.

By stopping the degradation in this way, the target nucleic acid is stabilized and the amount of protein translation of the target nucleic acid is increased.

Therefore, the present invention can provide:

A method for stabilizing a target nucleic acid, wherein an oligonucleotide changes the three-dimensional structure of the target nucleic acid through hybridization of a first nucleotide sequence and a second nucleotide sequence with the target nucleic acid, shortening the spatial distance of a total of four guanine repeat sequences on the target nucleic acid and the guanine repeat sequence on the oligonucleotide, and the four guanine repeat sequences form a guanine quadruple chain structure, thereby inhibiting the binding of a nuclease to the target nucleic acid or inhibiting the function of a nuclease, thereby stabilizing the target nucleic acid.

The oligonucleotide comprises: a nucleotide sequence portion containing 1 to 4 guanine repeat sequences on the target nucleic acid, a first nucleotide sequence and a second nucleotide sequence that hybridize with a nucleotide sequence near (i.e., on the 5' side or the 3' side) the guanine repeat sequence; and 3 to 0 guanine repeat sequences depending on the number of guanine repeat sequences on the target nucleic acid.

Regardless of whether it is a guanine quartet structure formed by a first-generation oligonucleotide or a guanine quartet structure formed by a second-generation oligonucleotide, the guanine quartet structure formed by these oligonucleotides produces the same target nucleic acid degradation inhibitory function when it is formed involving a guanine repeat sequence present in the 3' untranslated region (3'UTR) of the target nucleic acid.

[Chemistry 3]

(3) Ribosome diversion and partial sequence modification within a single target nucleic acid

As a result of the formation of a guanine quartet structure in the exon region of the target nucleic acid by the oligonucleotides of the present invention (including first-generation oligonucleotides and second-generation oligonucleotides), a loop is formed by the guanine quartet structure, so that when the ribosome bound to the target nucleic acid moves along the 3' direction, the guanine quartet structure portion is able to cross the target nucleic acid (ribosome diversion) and translate a protein in which the partial sequence of the target nucleic acid is changed.

Therefore, the present invention can provide:

A method for producing a modified protein from a target nucleic acid, wherein an oligonucleotide changes the three-dimensional structure of the target nucleic acid through hybridization of a first nucleotide sequence and a second nucleotide sequence with the target nucleic acid, thereby shortening the spatial distance between a total of four guanine repeat sequences on the target nucleic acid and the guanine repeat sequence on the oligonucleotide, and the four guanine repeat sequences form a guanine quadruple chain structure, thereby forming a loop portion of a part of the target nucleic acid, shunting ribosomes to the loop portion, and producing a modified protein from the target nucleic acid,

The oligonucleotide comprises: a nucleotide sequence portion containing 1 to 4 guanine repeat sequences on the target nucleic acid, a first nucleotide sequence and a second nucleotide sequence that hybridize with a nucleotide sequence near (i.e., on the 5' side or the 3' side) the above-mentioned guanine repeat sequence; and 3 to 0 guanine repeat sequences depending on the number of guanine repeat sequences on the target nucleic acid.

Regardless of whether it is a guanine quartet structure formed by a first-generation oligonucleotide or a guanine quartet structure formed by a second-generation oligonucleotide, when the guanine quartet structure formed by these oligonucleotides involves a guanine repeat sequence present in the exon region of the target nucleic acid, the function of transforming and translating a portion of the target nucleic acid sequence is similarly produced.

(4) Ribosome diversion between two target nucleic acids and fusion translation of partial sequences

The oligonucleotides of the present invention (including first-generation oligonucleotides and second-generation oligonucleotides) can also form a guanine quadruple chain structure between two target nucleic acids (sometimes referred to as the first target nucleic acid and the second target nucleic acid in this specification), and can substantially connect the two target nucleic acids. In the case where the nucleic acid formed by such connection is mRNA, when the mRNA is subjected to a ribosome reaction, the helicase activity does not work, and a reaction called ribosome shunting is performed by bypassing the stable nucleic acid higher-order structure. According to existing reports, the ribosome shunting phenomenon will not occur if a very long stem structure is not constructed. The guanine quadruple chain structure formed by the oligonucleotides of the present invention is a very stable nucleic acid higher-order structure, so a long stem structure is not required to induce ribosome shunting between two target nucleic acids.

When a guanine quartet structure is formed in the exon region of each of the two target nucleic acids, when the ribosome bound to the first target nucleic acid moves along the 3' direction, the ribosome crosses from the first target nucleic acid to the second target nucleic acid at the guanine quartet structure portion formed in the exon (ribosome diversion), and the translation of the partial sequence of the second target nucleic acid can be fused and translated subsequently to the translation of the partial sequence of the first target nucleic acid (Chemical 4).

Therefore, the present invention can provide:

A method for producing a fusion protein of a translation product of a partial sequence of a first target nucleic acid and a translation product of a partial sequence of a second target nucleic acid, wherein a first nucleotide sequence in an oligonucleotide hybridizes with a first target nucleic acid, a second nucleotide sequence hybridizes with a second target nucleic acid, and the oligonucleotide changes the three-dimensional structure of these target nucleic acids, so that the spatial distance of a total of four guanine repeat sequences, namely, 1 to 2 guanine repeat sequences on the first target nucleic acid, 1 to 2 guanine repeat sequences on the second target nucleic acid, and 2 to 0 guanine repeat sequences on the oligonucleotide is shortened, and these four guanine repeat sequences form a guanine quartet structure, thereby shunting ribosomes between the first target nucleic acid and the second target nucleic acid, thereby producing a fusion protein of a translation product of a partial sequence of the first target nucleic acid and a translation product of a partial sequence of the second target nucleic acid,

The oligonucleotide comprises:

A first nucleotide sequence that hybridizes with a nucleotide sequence near (i.e., on the 5' side or 3' side) the guanine repeat sequence on the first target nucleic acid,

A second nucleotide sequence that hybridizes with a nucleotide sequence near (i.e., 5' or 3' side of) the guanine repeat sequence to a nucleotide sequence portion containing 1 to 2 guanine repeat sequences on the second target nucleic acid, and

The number of guanine repeat sequences at 2 to 0 positions depends on the number of guanine repeat sequences on the first target nucleic acid and the number of guanine repeat sequences on the second target nucleic acid.

[Chemistry 4]

Uses of Staple Nucleic Acids of the Present Invention

The staple nucleic acid (oligonucleotide) of the present invention has the functions described in the item "Function of oligonucleotide", and therefore, by utilizing these functions, the present invention can provide a pharmaceutical composition comprising the staple nucleic acid of the present invention.

The pharmaceutical composition comprising the Staple nucleic acid of the present invention may be a method of administering a Staple nucleic acid prepared in vitro, or a method of administering a vector capable of producing the Staple nucleic acid to an organism to produce the Staple nucleic acid in vivo. Therefore, the pharmaceutical composition of the present invention may be prepared by formulating a therapeutically effective amount of the Staple nucleic acid of the present invention alone or together with a pharmaceutically acceptable carrier (additive), excipient and/or diluent, or, alternatively, a vector capable of producing a therapeutically effective amount of the Staple nucleic acid of the present invention in vivo may be formulated alone or together with a pharmaceutically acceptable carrier (additive), excipient and/or diluent.

Here, the term "therapeutically effective amount" used in the present specification refers to an amount of the Staple nucleic acid of the present invention that is effective for obtaining a therapeutic effect based on a desired primary action, regardless of the presence or absence of side effects.

The "pharmaceutically acceptable carrier" used in this specification refers to a pharmaceutically acceptable carrier commonly used in the art, and refers to a carrier suitable for use in human and animal tissues without excessive toxicity, irritation, allergic reaction or other problems and without complications. Such carriers include pharmaceutically acceptable materials, compositions or carriers that participate in transporting or delivering the target compound from an organ or part of the body to another organ or part of the body, such as liquid or solid fillers, diluents, excipients, manufacturing aids (such as lubricants, talc, magnesium, calcium stearate, zinc stearate or stearic acid) or solvents containing materials, but are not limited to these.

The pharmaceutical composition containing the staple nucleic acid of the present invention can be used in combination with other components. Such other components are not particularly limited and can be used in combination with any components.

The staple nucleic acid (oligonucleotide) of the present invention also has the functions described in the "Function of oligonucleotide" section, and a kit for achieving these functions comprising the staple nucleic acid of the present invention can be provided. That is, the following can be provided:

A kit for inhibiting protein expression comprising a staple nucleic acid for achieving the function of "(1) inhibiting protein expression" described above;

A kit for stabilizing a target nucleic acid comprising a staple nucleic acid for achieving the function of "(2) stabilization of a target nucleic acid" described above;

A kit for producing a modified protein of a partial sequence of a target nucleic acid containing a staple nucleic acid for realizing the function of "(3) ribosome diversion and modified translation of a partial sequence in a single target nucleic acid" as described above,

A kit for producing a fusion protein of partial sequences of two target nucleic acids containing a staple nucleic acid for realizing the function of "(4) ribosome diversion and fusion translation of partial sequences between two target nucleic acids" as described above. The contents of the kit are expanded as the term of the staple nucleic acid of the present invention is expanded.

For example, as a more specific embodiment, a kit for inhibiting protein expression comprising a staple nucleic acid for achieving the function of "(1) inhibiting protein expression" described above may be provided:

A protein expression inhibition kit comprising a G-donating oligonucleotide,

The G-donating oligonucleotide comprises: a first nucleotide sequence and a second nucleotide sequence that hybridize with a nucleotide sequence near (i.e., on the 5' side or the 3' side) the guanine repeat sequence for a nucleotide sequence portion containing 1 to 3 guanine repeat sequences on the target nucleic acid; and 3 to 1 guanine repeat sequence depending on the number of guanine repeat sequences on the target nucleic acid,

The G-donating oligonucleotide can change the three-dimensional structure of the target nucleic acid by hybridizing the first nucleotide sequence and the second nucleotide sequence with the target nucleic acid, thereby shortening the spatial distance between the guanine repeat sequences on the target nucleic acid and the guanine repeat sequences on the G-donating oligonucleotide, which together form a guanine quadruple chain structure, thereby inhibiting the binding of the ribosome to the target nucleic acid or inhibiting the function of the ribosome.

The present invention is specifically disclosed by way of examples below. The present invention is not limited to the examples shown below in any manner.

Example

Example 1: Design of the second generation Staple nucleic acid

In this example, a second generation staple nucleic acid was prepared in which a guanine quartet structure was formed by supplying a guanine repeat sequence using an oligonucleotide with several mRNAs as targets.

(1) Second generation Staple nucleic acid with 1+1100nt RNA as target nucleic acid

In this experiment, a model nucleic acid RNA sequence (1+1100 nt RNA) was designed and used in which one guanine repeat sequence was present adjacent to both ends of a 100-base loop sequence. The base sequence of the 1+1100 nt RNA as the target nucleic acid is as follows.

【Chemistry 5】

This sequence is a sequence having two guanine repeating sequences in the target nucleic acid, and can form a guanine quartet structure only when two guanine repeating sequences are supplied by the second-generation Staple nucleic acid of the present invention.

The following oligonucleotides were designed and prepared for the target nucleic acid. When designing the oligonucleotides, an attempt was made to design type I oligonucleotides and type II oligonucleotides based on the positional relationship between the binding site of the first nucleotide sequence and the binding site of the second nucleotide sequence on the target nucleic acid and the guanine repeat sequence on the target nucleic acid. That is, the type I oligonucleotide is arranged in a manner that the two guanine repeat sequences are located between the first nucleotide sequence and the second nucleotide sequence, and the type II oligonucleotide is arranged and configured in a manner that the two guanine repeat sequences sandwich the first nucleotide sequence and the second nucleotide sequence.

As type I oligonucleotides, we designed and synthesized:

(1-1) G-supply type staple nucleic acid (for 1+1100nt RNA, type I): Type I G-supply type staple nucleic acid with 1+1100nt RNA as target nucleic acid

(1-2) Staple_A (for 1+1100nt RNA, type I): The guanine repeat sequence of the oligonucleotide in (1-1) is replaced with an adenine repeat sequence.

Oligonucleotides used in this specification were purchased from Thermo Fisher Scientific, Inc. In addition, all samples were used after desalting and purification.

【Chemistry 6】

In addition, as type II oligonucleotides, we designed and prepared:

(1-3) G-supplying staple nucleic acid (type II): Type II G-supplying staple nucleic acid with 1+1100 nt RNA as target nucleic acid

(1-4) Staple_A (type II): A oligonucleotide in (1-3) in which one of the guanine repeat sequence portions is replaced with an adenine repeat sequence.

【Chemistry 7】

In order to evaluate the function of G-donating staple nucleic acid in constructing a guanine quartet structure of a target nucleic acid using these oligonucleotides, fluorescence measurement using thioflavin T (ThT), which is known to bind to a guanine quartet structure and emit fluorescence, was performed.

For the RNA sample of sequence number 1, the sample was prepared under the following conditions, and the fluorescence measurement using thioflavin T (ThT) was performed by fluorescence luminescence measurement using ThT. A mixed solution (total volume: 50 μl) of RNA sample (2 μM), KCl (100 mM), 20 mM Tris-HCl buffer (pH 7.6), and G-supply type Staple nucleic acid (2.5 μM) was heated at 90°C for 2 minutes and cooled to 20°C at 1°C per minute. Thereafter, 10 μl (total volume: 60 μl) of 3 μM ThT solution was added to the mixed solution at a final concentration of 0.5 μM, and incubated at room temperature for 30 minutes. The sample was excited with 440 nm light using FP-8500 spectrofluorometer (JASCO, Inc.), and the fluorescence signal at 487 nm was measured. The measurement was performed by measuring the wavelength of 450 nm to 600 nm, the scanning speed of 200 nm/min, and accumulating 3 times.

The results of fluorescence measurement using oligonucleotides (1-1) and (1-2) of type I are shown in (A) of Figure 1. That is, in the presence of the G-donating staple nucleic acid (1-1), a strong fluorescence signal derived from ThT was obtained, while in the presence of the staple nucleic acid (1-2) (Staple_A) in which the guanine repeat sequence was mutated to adenine in order to prevent the formation of a guanine quartet structure, there was no significant change in the fluorescence signal (Figure 1 (A)).

The results of fluorescence measurement using the type II oligonucleotide (1-3) and the type II oligonucleotide (1-4) are shown in (B) of Figure 1. That is, in the presence of the G-donating staple nucleic acid (1-3), the fluorescence signal was weaker than that of the type I oligonucleotide, but a fluorescence signal derived from ThT was obtained. On the other hand, in the presence of the staple nucleic acid (1-4) (Staple_A) in which the guanine repeat sequence was mutated to adenine in order to prevent the formation of a guanine quartet structure, there was no significant change in the fluorescence signal (Figure 1 (B)).

These results indicate that both oligonucleotide (1-1) and oligonucleotide (1-3) function as G-donating staple nucleic acids. In addition, the fluorescence signal differs depending on the insertion position of the guanine repeat sequence in the G-donating staple nucleic acid, and a stronger fluorescence signal is obtained by inserting the guanine repeat sequence centered on the guanine repeat sequence (Figure 1), indicating that the efficiency of forming the guanine quadruple chain structure depends on the binding mode of the G-donating staple nucleic acid.

(2) Second generation Staple nucleic acid with 2+1100 nt RNA as target nucleic acid

In this experiment, "2+1100 nt RNA" was designed and used as a model target nucleic acid. The base sequence of the 2+1100 nt RNA as the target nucleic acid is as follows.

【Chemistry 8】

This sequence is a sequence having three guanine repeating sequences in the target nucleic acid, and can form a guanine quartet structure only when one guanine repeating sequence is supplied by the second-generation Staple nucleic acid of the present invention.

The following oligonucleotides were designed and prepared for the target nucleic acid. When designing the oligonucleotides, an attempt was made to design the oligonucleotides based on the positional relationship between the binding sites of the first nucleotide sequence and the second nucleotide sequence on the target nucleic acid and the guanine repeat sequence on the target nucleic acid. That is, the oligonucleotides of this embodiment are configured in a manner such that one guanine repeat sequence is located between the first nucleotide sequence and the second nucleotide sequence.

As oligonucleotides, we designed and prepared:

(2-1) G-supply type staple nucleic acid (for 2+1100nt RNA): G-supply type staple nucleic acid with 2+1100nt RNA as target nucleic acid

(2-2) Staple_A (for 2+1100 nt RNA): The guanine repeat sequence of the oligonucleotide in (2-1) was replaced with an adenine repeat sequence.

【Chemistry 9】

In order to evaluate the function of G-donating staple nucleic acid in constructing a guanine quartet structure of a target nucleic acid using these oligonucleotides, fluorescence measurement using thioflavin T (ThT), which is known to bind to a guanine quartet structure and emit fluorescence, was performed.

Fluorescence measurement using thioflavin T (ThT) was performed by the above-mentioned method.

The results of fluorescence measurement using the oligonucleotides (2-1) and (2-2) are shown in (A) of Figure 2. That is, in the presence of the G-donating staple nucleic acid (2-1), a strong fluorescence signal derived from ThT was obtained, while in the presence of the staple nucleic acid (Staple_A) (2-2) in which the guanine repeat sequence was mutated to adenine in order to prevent the formation of a guanine quartet structure, there was no significant change in the fluorescence signal (Figure 2 (A)).

This result shows that the oligonucleotide (2-1) functions as a G-donating staple nucleic acid.

(3) Second generation Staple nucleic acid with 1+1100nt_mut 1RNA as target nucleic acid

In this experiment, another model target nucleic acid, "1+1100nt_mut 1RNA", was designed and used. The base sequence of the target nucleic acid, 1+1100nt_mut 1RNA, is as follows.

【Chemistry 10】

This sequence is a sequence having two guanine repeating sequences in the target nucleic acid, and can form a guanine quartet structure only when two guanine repeating sequences are supplied by the second-generation Staple nucleic acid of the present invention.

The following oligonucleotides were designed and prepared for the target nucleic acid. When designing the oligonucleotides, an attempt was made to design the oligonucleotides based on the positional relationship between the binding sites of the first nucleotide sequence and the second nucleotide sequence on the target nucleic acid and the guanine repeat sequence on the target nucleic acid. That is, the oligonucleotides of this embodiment are configured in such a way that two guanine repeat sequences are located between the first nucleotide sequence and the second nucleotide sequence.

As oligonucleotides, we designed and prepared:

(3-1) G-donating staple nucleic acid (for 1+1100nt_mut 1RNA): G-donating staple nucleic acid with 1+1100nt_mut 1RNA as target nucleic acid

(3-2) Staple_A (for 1+1100nt_mut 1 RNA): The guanine repeat sequence portion of the oligonucleotide of (3-1) was replaced with an adenine repeat sequence.

【Chemistry 11】

In order to evaluate the function of G-donating staple nucleic acid in constructing a guanine quartet structure of a target nucleic acid using these oligonucleotides, fluorescence measurement using thioflavin T (ThT), which is known to bind to a guanine quartet structure and emit fluorescence, was performed.

Fluorescence measurement using thioflavin T (ThT) was performed by the above-mentioned method.

The results of fluorescence measurement using the oligonucleotides (3-1) and (3-2) are shown in (B) of Figure 2. That is, in the presence of the G-donating Staple nucleic acid (3-1), a strong fluorescence signal derived from ThT was obtained, while in the presence of the Staple nucleic acid (3-2) (Staple_A) in which the guanine repeat sequence was mutated to adenine in order to prevent the formation of a guanine quartet structure, there was no significant change in the fluorescence signal (Figure 2 (B)).

This result shows that the oligonucleotide (3-1) functions as a G-donating staple nucleic acid.

(4) Second generation Staple nucleic acid with 1+1100nt_mut 2RNA as target nucleic acid

In this experiment, another "1+1100nt_mut 2RNA" was designed and used as a model target nucleic acid. The base sequence of the 1+1100nt_mut 2RNA as the target nucleic acid is as follows.

【Chemistry 12】

This sequence is a sequence having two guanine repeating sequences in the target nucleic acid, and can form a guanine quartet structure only when two guanine repeating sequences are supplied by the second-generation Staple nucleic acid of the present invention.

The following oligonucleotides were designed and prepared for the target nucleic acid. When designing the oligonucleotides, an attempt was made to design the oligonucleotides based on the positional relationship between the binding sites of the first nucleotide sequence and the second nucleotide sequence on the target nucleic acid and the guanine repeat sequence on the target nucleic acid. That is, the oligonucleotides of this embodiment are configured in such a way that two guanine repeat sequences are located between the first nucleotide sequence and the second nucleotide sequence.

As oligonucleotides, we designed and prepared:

(4-1) G-donating staple nucleic acid (for 1+1100nt_mut 2RNA): G-donating staple nucleic acid with 1+1100nt_mut 2RNA as target nucleic acid

(4-2) Staple_A (for 1+1100nt_mut 2RNA): The guanine repeat sequence of the oligonucleotide of (4-1) was replaced with an adenine repeat sequence.

【Chemistry 13】

In order to evaluate the function of G-donating staple nucleic acid in constructing a guanine quartet structure of a target nucleic acid using these oligonucleotides, fluorescence measurement using thioflavin T (ThT), which is known to bind to a guanine quartet structure and emit fluorescence, was performed.

Fluorescence measurement using thioflavin T (ThT) was performed by the above-mentioned method.

The results of fluorescence measurement using the oligonucleotides (4-1) and (4-2) are shown in (C) of Figure 2. That is, in the presence of the G-donating Staple nucleic acid (4-1), a strong fluorescence signal derived from ThT was obtained, while in the presence of the Staple nucleic acid (Staple_A) (4-2) in which the guanine repeat sequence was mutated to adenine in order to prevent the formation of a guanine quartet structure, there was no significant change in the fluorescence signal (Figure 2 (C)).

This result shows that the oligonucleotide (4-1) functions as a G-donating staple nucleic acid.

(5) Second generation Staple nucleic acid with 1+1100nt RNA-2 as target nucleic acid

In this experiment, another model target nucleic acid "1+1100nt RNA-2" was designed and used. The base sequence of the target nucleic acid 1+1100nt RNA-2 is as follows.

【Chemistry 14】

This sequence is a sequence having two guanine repeating sequences in the target nucleic acid, and can form a guanine quartet structure only when two guanine repeating sequences are supplied by the second-generation Staple nucleic acid of the present invention.

The following oligonucleotides were designed and prepared for the target nucleic acid. When designing the oligonucleotides, an attempt was made to design the oligonucleotides based on the positional relationship between the binding sites of the first nucleotide sequence and the second nucleotide sequence on the target nucleic acid and the guanine repeat sequence on the target nucleic acid. That is, the oligonucleotides of this embodiment are configured in such a way that two guanine repeat sequences are located between the first nucleotide sequence and the second nucleotide sequence.

As oligonucleotides, we designed and prepared:

(5-1) G-supplying staple nucleic acid (for 1+1100nt RNA-2): G-supplying staple nucleic acid with 1+1100nt RNA-2 as the target nucleic acid

(5-2) Staple_A (for 1+1100nt RNA-2): The guanine repeat sequence of the oligonucleotide of (5-1) is replaced with an adenine repeat sequence.

【Chemistry 15】

(6) Second generation Staple nucleic acid with TRPC65'UTR RNA as target nucleic acid

In this experiment, a staple nucleic acid for another target nucleic acid, "TRPC65'UTR RNA", was designed and used. The base sequence of the staple nucleic acid for the target nucleic acid, TRPC65'UTR RNA, is as follows.

【Chemistry 16】

This sequence is a sequence having two guanine repeating sequences in the target nucleic acid, and can form a guanine quartet structure only when two guanine repeating sequences are supplied by the second-generation Staple nucleic acid of the present invention.

The following oligonucleotides were designed and prepared for the target nucleic acid. When designing the oligonucleotides, an attempt was made to design the oligonucleotides based on the positional relationship between the binding sites of the first nucleotide sequence and the second nucleotide sequence on the target nucleic acid and the guanine repeat sequence on the target nucleic acid. That is, the oligonucleotides of this embodiment are configured in such a way that two guanine repeat sequences are located between the first nucleotide sequence and the second nucleotide sequence.

As oligonucleotides, we designed and prepared:

(6-1) G-donating staple nucleic acid_T (1-1-1) (for TRPC6 5'UTR RNA): A G-donating staple nucleic acid with TRPC6 5'UTR RNA as the target nucleic acid and the arrangement of T near the guanine repeat sequence being 1 T-G repeat sequence-1 T-G repeat sequence-1 T

(6-2) G-donating staple nucleic acid_T (2-1-2) (for TRPC6 5'UTR RNA): A G-donating staple nucleic acid with TRPC6 5'UTR RNA as the target nucleic acid and the configuration of T near the guanine repeat sequence being 2 T-G repeat sequences-1 T-G repeat sequence-2 Ts

(6-3) G-supplying staple nucleic acid_T (3-1-3) (for TRPC6 5'UTR RNA): A G-supplying staple nucleic acid with TRPC6 5'UTR RNA as the target nucleic acid and the configuration of T near the guanine repeat sequence being 3 T-G repeat sequences-1 T-G repeat sequence-3 Ts

(6-4) G-supplying staple nucleic acid_T (1-2-1) (for TRPC6 5'UTR RNA): A G-supplying staple nucleic acid with TRPC6 5'UTR RNA as the target nucleic acid and the configuration of T near the guanine repeat sequence being 1 T-G repeat sequence-2 T-G repeat sequences-1 T

(6-5) G-donating staple nucleic acid_T (1-3-1): A G-donating staple nucleic acid with TRPC6 5'UTR RNA as the target nucleic acid and the arrangement of T near the guanine repeat sequence being 1 T-G repeat sequence-3 T-G repeat sequences-1 T

(6-6) G-donating staple nucleic acid_T (2-2-2): A G-donating staple nucleic acid with TRPC6 5'UTR RNA as the target nucleic acid and the arrangement of T near the guanine repeat sequence being 2 T-G repeat sequences-2 T-G repeat sequences-2 T

(6-7) G-donating Staple Nucleic Acid_T(2-1-2)_36mer: shortened by 2 bases from the 5' end and 2 bases from the 3' end of the oligonucleotide (6-2)

(6-8) G-donating Staple Nucleic Acid_T(2-1-2)_32mer: shortened by 4 bases from the 5' end and 4 bases from the 3' end of the oligonucleotide (6-2)

(6-9) G-donating Staple Nucleic Acid_T(2-1-2)_28mer: The oligonucleotide of (6-2) is shortened by 6 bases from the 5' end and 6 bases from the 3' end. These Staple Nucleic Acids are used in the stop test.

【Chemistry 17】

(7) Second generation Staple nucleic acid with TRPC6 5'UTR RNA as target nucleic acid

In this experiment, a staple nucleic acid for another target nucleic acid, "TRPC65'UTR RNA", was designed and used. The base sequence of the staple nucleic acid for the target nucleic acid, TRPC65'UTR RNA, is as follows.

【Chemistry 18】

This sequence is a sequence having two guanine repeating sequences in the target nucleic acid, and can form a guanine quartet structure only when two guanine repeating sequences are supplied by the second-generation Staple nucleic acid of the present invention.

The following oligonucleotides were designed and prepared for the target nucleic acid. When designing the oligonucleotides, an attempt was made to design the oligonucleotides based on the positional relationship between the binding sites of the first nucleotide sequence and the second nucleotide sequence on the target nucleic acid and the guanine repeat sequence on the target nucleic acid. That is, the oligonucleotides of this embodiment are configured in such a way that two guanine repeat sequences are located between the first nucleotide sequence and the second nucleotide sequence.

As oligonucleotides, we designed and prepared:

(7-1) G-donating staple nucleic acid (for TRPC6 5'UTR RNA): A G-donating staple nucleic acid using TRPC6 5'UTR RNA as a target nucleic acid. This staple nucleic acid is used in an in vitro translation experiment.

【Chemistry 19】

Example 2: Study on the formation site of guanine quadruple chain structure of second generation Staple nucleic acid on target nucleic acid

In this example, a stop test was performed for each of the staple nucleic acids prepared in (5) and (6) of Example 1 to confirm at which site on the target nucleic acid a guanine quartet structure was formed.

Reverse transcription can generate full-length complementary DNA (cDNA) from RNA. On the other hand, if reverse transcription is performed on RNA with a guanine quartet structure, the cDNA extension reaction of the reverse transcriptase will be inhibited by the guanine quartet structure, generating short cDNA. By confirming this, it is possible to confirm where the target nucleic acid has formed a guanine quartet structure.

In the stop test, the determination of the position of the guanine quartet structure by reverse transcription and the determination of the target DNA sequence by the Sanger method are performed simultaneously, thereby making it possible to understand where in the sequence the reverse transcription stops.

In the stop test using the "(5-1) G-donating staple nucleic acid (for 1+1100nt RNA-2)" (SEQ ID NO: 16) and "(5-2) Staple_A (for 1+1100nt RNA-2)" (SEQ ID NO: 17) prepared in "(5) Second generation staple nucleic acid using 1+1100nt RNA-2 as target nucleic acid" of Example 1, the template RNA "1+1100nt RNA-2" (SEQ ID NO: 15) (0.3 μM) during the reverse transcription reaction, the G-donating staple nucleic acid (5-1) (SEQ ID NO: 16) (1.0 μM), KCl (100 mM), Tris-HCl buffer (pH 7.6, 50 mM), 5'-CD4 labeled 3'-DNA primer [5'-CD4-CAC ACA GGA AAC AGC TAT GAC CAT GAT A mixed solution of TA-3'] (SEQ ID NO: 30) (0.3 μM) was heated at 90°C for 2 minutes and cooled to 20°C at a rate of 1°C per minute. After that, a reverse transcription reaction solution of the annealed sample, Rever Tra Ace reverse transcriptase (TOYOBO, Inc.) (100 units), MgCl ₂ (5 mM), and dNTPs (0.86 mM) was reacted at 42°C for 30 minutes, and then 60 units of RNase H were added and reacted at 37°C for 30 minutes. The resulting final reaction solution was analyzed using a capillary sequencer CEQ8000 (BECKMAN COULTER, Inc.). As a sequencing marker, DNA size marker-600 (BECKMAN COULTER, Inc.) was used. As a negative control, a stop test was performed using Staple_A nucleic acid (5-2) (SEQ ID NO: 17) instead of G-donating Staple nucleic acid (5-1) (SEQ ID NO: 16) according to the same protocol.

In the stop test of "(6-1) G-supplying staple nucleic acid_T(1-1-1) (for TRPC6 5'UTR RNA)" (sequence number 19) prepared in the "(6) 2nd generation staple nucleic acid using TRPC6 5'UTR RNA as target nucleic acid" of Example 1 for "TRPC6 5'UTR RNA" (sequence number 18), the template RNA "TRPC6 5'UTR RNA" (sequence number 18) (0.3μM) during the reverse transcription reaction, G-supplying staple nucleic acid (1.0μM), and 5'-CD4 labeled 3'-DNA primer [5'-CD4-GAG ATT TCC AAC TAT GTA TAC CTG-3'] (sequence number 31) (0.3μM) were used, and the stop test was performed by the same steps as the above-mentioned stop test. As a negative control, a Staple_A nucleic acid in which the G repeat sequence was replaced with an equal number of A was used instead of the G-donating Staple nucleic acid (6-1) (SEQ ID NO: 19), and the stop test was performed in the same manner.

The results of these stop tests are shown in Figure 3. When "1+1 100nt RNA-2" (SEQ ID NO: 15) was used as the target nucleic acid, the RTase stop signal derived from the guanine quartet structure was detected in the presence of the G-donating Staple nucleic acid (5-1), and the RTase stop signal derived from the guanine quartet structure was not detected in the presence of Staple_A (5-2) (Figure 3 (A)).

When the G-donating staple nucleic acid (6-1) was used as the target nucleic acid for the 5'UTR of the TRPC6 gene ("TRPC6 5'UTR RNA" (SEQ ID NO: 18)), an RTase stop signal was detected in the presence of the G-donating staple nucleic acid ( FIG. 3(B) ).

These results indicate that a guanine quartet structure can be constructed using a G-donating staple nucleic acid not only in the case of the model sequence (1+1 100 nt RNA) ( FIG. 3(A) ) but also in the case of the gene (TRPC6) ( FIG. 3(B) ).

In this Example, in order to investigate (A) the effect of the number of T linkers in a G-donating staple nucleic acid on the function of the staple nucleic acid, and (B) the effect of changing the length of the G-donating staple nucleic acid itself on the function of the staple nucleic acid, a stop test was performed by the same procedure as the above procedure in the stop test using the G-donating staple nucleic acids (6-1) to (6-6) and (6-10) to (6-12) in (A) using "TRPC6 5'UTR RNA" (SEQ ID NO: 18) as the target nucleic acid (SEQ ID NO: 19 to SEQ ID NO: 24, SEQ ID NO: 38 to SEQ ID NO: 40, respectively), and (6-2) (SEQ ID NO: 20) in (B) and the G-donating staple nucleic acids (6-7) to (6-9) in which the lengths of the first nucleotide sequence and the second nucleotide sequence of (6-2) were shortened (SEQ ID NO: 25 to SEQ ID NO: 27, respectively).

It can be seen that when the length of the T linker between the target nucleic acid recognition sequence and the guanine repeat sequence in the G-supplying staple nucleic acid is changed, the reverse transcription reaction of RTase is inhibited when the T linker is any one of 0 to 3 structures (Figure 4 (A)). In addition, when the length of the target nucleic acid recognition region, i.e., the first nucleotide sequence and the second nucleotide sequence, is shortened, the RTase stop signal cannot be detected when it is shorter than 32 bases (Figure 4 (B)).

These results indicate that the reverse transcription inhibitory effect on Rtase does not depend on the length of the linker between the G repeat sequences of the G-donating staple nucleic acid, but depends on the stability (length) of the double strand formed between the G-donating staple nucleic acid and the target nucleic acid.

Example 3: Study on the protein expression inhibition effect of the second generation Staple nucleic acid

In this example, the second generation Staple nucleic acid prepared in Example 1 was used to confirm whether it produced a real-time protein expression inhibitory effect.

As target nucleic acid and G-donating staple nucleic acid for it, we used:

The "(7-1) G-supplying staple nucleic acid (for TRPC6 5'UTR RNA)" (sequence number 29) prepared in Example 1 "(7) Second-generation staple nucleic acid with TRPC6 5'UTR RNA as target nucleic acid" for "TRPC6 5'UTR RNA" (sequence number 28) was used to evaluate the effect of the guanine quadruple chain structure constructed by the G-supplying staple nucleic acid in (7) TRPC65'UTR RNA on the protein translation reaction by an in vitro translation experiment.

Regarding the effect on the protein translation reaction of each target nucleic acid, two reporter genes (linked to the target nucleic acid) were used: firefly luciferase (FL) that is Cap-dependent for translation and Renilla luciferase (RL) that is IRES-dependent for translation (summary shown in (A) of FIG. 5 ). By comparing the luminescent signals from FL and RL, the effect of the constructed guanine quadruple chain structure on gene expression can be quantitatively evaluated.

In the evaluation based on in vitro translation, each target nucleic acid was used as an mRNA template (1 μg), and 20 μL of a cell-free protein expression mixture containing 0.14 μM or no G-supplying staple nucleic acid (RTS100 Wheat Germ CECF Kit, 5 Prime, Inc) was reacted at 24°C for 150 minutes. Luciferase activity was measured using a luciferase assay system (Promega, Inc.) and a Renilla luciferase assay system (Promega, Inc.) and a POWERSCAN H1 microplate reader (BioTek, Inc.).

As a result of in vitro translation, in the presence of a G-donating staple nucleic acid, the translation efficiency of the transcripts having each gene sequence decreased ( FIG. 5(B) ). These results indicate that the use of a G-donating staple nucleic acid can inhibit the expression of a target gene in vitro.

Example 4: Study on the target nucleic acid stabilization effect of the Staple nucleic acid of the present invention

In this example, the first-generation Staple nucleic acid and the second-generation Staple nucleic acid were used to confirm whether the target nucleic acid stabilization effect was produced.

The research team of the inventors has previously successfully developed a compound RGB-1 that can selectively bind and stabilize the guanine quartet structure (JACS, 2016). Some genes whose protein expression levels increase when RGB-1 is added to cells were found. Furthermore, when exploring the guanine quartet structure using a stop test that can determine the position where the guanine quartet structure is formed, it was found that RGB-1 induces the formation of the guanine quartet structure at the 3'UTR of a specific mRNA.

Therefore, in this embodiment, it is thought that by making a guanine quadruple chain structure present in the target nucleic acid, it is possible to block the exonuclease by physical means to obtain resistance, and a Staple nucleic acid targeting the 3'UTR region of the target nucleic acid is designed and produced in order to improve the stability of the target nucleic acid (and thereby increase protein expression).

In this example, a target nucleic acid (SEQ ID NO: 32) of the following sequence was designed and used. In this example, a staple nucleic acid acting on the target nucleic acid was prepared as follows.

【Chemistry 20】

Using the prepared template RNA as the target nucleic acid, the template RNA and the Staple nucleic acid were incubated at 90°C for 2 minutes in a composition of KCl (150mM) and Tris-HCl buffer (pH7.6) (20mM), and annealed by decreasing the temperature to 20°C at 1°C/min (template RNA: Staple = 1: 1.25). Using the annealing solution, a measurement solution (total volume: 150μl) was prepared in the form of RNA (0.04μM), 1×RNaseR buffer, RNase R (0.01units) or milliQ (control). Using a UV/VIS Spectrophotometer V-560 (JASCO), the absorption spectrum at 260nm was measured at 37°C for 1 hour. This was repeated 3 times to make N=3. In this step, the UV absorption value increases when the target nucleic acid is decomposed, thereby enabling the detection of the decomposition of the target nucleic acid.

As a result of inducing the formation of a guanine quadruple chain structure in the 3'UTR of the target nucleic acid by introducing a staple nucleic acid, the stability of the target nucleic acid was significantly improved even in the presence of RNaseR (Figure 6). Therefore, the presence of a staple nucleic acid significantly improves the stability. As a result, the expression level of the protein is increased.

Example 5: Study on the ribosome diversion effect of the Staple nucleic acid of the present invention in a single target nucleic acid

In this example, the first generation Staple nucleic acid was used to confirm the occurrence of ribosome diversion effect on a partial sequence of a single target nucleic acid.

It is well known that when the ribosome reaction is hindered by a high-order structure with high thermal stability and bulky volume, a ribosome shunting strategy is adopted to skip (bypass) the structure and continue protein translation. In this embodiment, a technology for controlling ribosome shunting and bypassing the stop codon in a single target nucleic acid is established.

In this example, the target nucleic acid of the following sequence was designed and used (PTC GFP is sequence number 34, Δtype1 GFP is sequence number 35). The mRNA of PTC GFP has the following characteristics: "TGA" between the first nucleotide sequence and the second nucleotide sequence shown in the underlined portion functions as a stop codon, and translation stops at this codon.

【Chemistry 21

In this example, a Staple nucleic acid acting on the target nucleic acid was prepared as follows. Here, the Staple nucleic acid is of the first generation, and both Staple nucleic acid B1 and Staple nucleic acid B2 can bind to the target nucleic acid of PTC GFP, but not to Δtype1 GFP. Both Staple nucleic acids are designed to be able to cause ribosome diversion by skipping the stop codon. It should be noted that the 3' end side of Staple nucleic acid B1 is shortened by 3 bases (GCT) compared with Staple nucleic acid B2.

【化22】

Using the mRNA transcribed from the above sequence PTC GFP (SEQ ID NO. 34) or Δtype1 GFP (SEQ ID NO. 35) as a template (1 μg), 20 μL of a cell-free protein expression mixture (RTS100 Wheat Germ CECF Kit, 5 Prime, Inc) containing 0.14 μM of each Staple nucleic acid or not was reacted at 24°C for 150 minutes. The obtained RNA was used as an mRNA template (1 μg), and 20 μL of a cell-free protein expression mixture (RTS100 Wheat Germ CECF Kit, 5 Prime, Inc) containing 0.14 μM of Staple nucleic acid or not was reacted at 24°C for 150 minutes. The excitation wavelength was set to 480 nm, and fluorescence was measured using a fluorescence spectrophotometer (Jasco FP-8500) at 25°C.

The results are shown in Figure 7. In this figure, the GFP signal intensity when GFP mRNA is expressed is set to 100, and the GFP signal intensities of PTCGFP (no staple nucleic acid), Δtype1 GFP (no staple nucleic acid), PTC GFP (Staple nucleic acid B1), and PTC GFP (Staple nucleic acid B2) are compared. As a result, the signal intensity of PTC GFP (no staple nucleic acid) is 3% of that of GFP, while the intensities of PTC GFP (Staple nucleic acid B1) and PTC GFP (Staple nucleic acid B2) are increased to about 37% and 8%, respectively.

Example 6: Study on the target nucleic acid stabilization effect of the first generation of Staple nucleic acid of the present invention

In this example, the first generation Staple nucleic acid was used to confirm whether a stabilization effect occurs when mRNA is used as the target nucleic acid and whether protein expression is increased as a result.

(6-1) Stabilization of mRNA

As described in Example 4, in a cell-free system, the first-generation Staple nucleic acid of the present invention can play a role in stabilizing target nucleic acids.

In this example, the target nucleic acid "Random sequence_2+2 100nt" (a target nucleic acid having a sequence formed by connecting a random sequence and 2+2 100nt) (SEQ ID NO: 41) of the following sequence was designed and used. In addition, as a control, "Random sequence_2+2 100nt MutA" (SEQ ID NO: 42) was designed in which the 4 guanine repeats of the target nucleic acid "Random sequence_2+2 100nt" were changed to "AAA".

【Chemistry 23】

In this example, the first generation of staple nucleic acid acting on the target nucleic acid was further prepared as follows: In the case of this structure, it was designed so that a guanine quartet structure was formed in the region corresponding to the 3'UTR of the target nucleic acid by the staple nucleic acid.

【Chemistry 24】

Using the prepared template RNA as the target nucleic acid, the template RNA and the Staple nucleic acid were incubated at 90°C for 2 minutes in a composition of KCl (150mM) and Tris-HCl buffer (pH7.6) (10mM), and annealed by decreasing the temperature to 20°C at 1°C/min (template RNA: Staple = 1: 1.25). Using the annealing solution, a measurement solution (total volume: 150μl) was prepared in the form of RNA (0.04μM), 1×RNaseR buffer, RNase R (0.1units) or milliQ (control). Using a UV/VIS Spectrophotometer V-560 (JASCO), the absorption at 260nm was measured at 37°C for 1 hour. This was repeated 3 times and N=3. In this step, the UV absorption value increases when the target nucleic acid is decomposed, thereby enabling the detection of the decomposition of the target nucleic acid.

By introducing Staple nucleic acid, a guanine quartet structure is induced to form on the 3' end side of the target nucleic acid "random sequence_2+2100nt", and as a result, in the presence of RNaseR, the resistance of the target nucleic acid to 3'→5' exonuclease activity is improved, and it can be confirmed that the stability of the target nucleic acid is significantly improved (Figure 8-1, upper figure). On the other hand, in the case of "random sequence_2+2100nt MutA" that does not contain guanine repeats, a guanine quartet structure is not formed, so the decomposition of "random sequence_2+2100nt MutA" caused by 3'→5' exonuclease activity in the presence of RNaseR is carried out to the same extent as the negative control ("w/oligo" group) (Figure 8-1, lower figure). Therefore, the presence of Staple nucleic acid significantly improves stability.

(6-2) Effect of increasing protein expression

In this example, it was confirmed whether the staple nucleic acid of the present invention can form a guanine quartet structure in the target nucleic acid in cells and whether the stability of the target nucleic acid can be improved (and thus the protein expression can be increased) in cells as a result.

In this example, a target nucleic acid of the following sequence "Firefly_2+2" (a 2+2 sequence connected to the gene sequence of firefly luciferase) (SEQ ID NO: 44) was designed and used.

【Chemistry 25】

In this example, a first generation staple nucleic acid acting on the target nucleic acid was prepared as follows: Its structure was designed so that a guanine quartet structure was formed in the region corresponding to the 3'UTR of the target nucleic acid by the staple nucleic acid.

【Chemistry 26】

HEK 293T cell#16 cells were seeded in a 96-well plate at a cell density of 2×10 ⁴ cells/ml and cultured for 72 hours in 100 μl of D-MEM medium containing 10% FBS and 1% penicillin. When the cells were 80% confluent, the medium was replaced with 100 μl of D-MEM medium containing 10% FBS and cultured for 3 hours.

The cells were divided into two groups. For the group using Staple nucleic acid, pSuper expressing the Staple nucleic acid sequence under the control of the H1 promoter was used. For the group not using Staple nucleic acid, 1 μg of an empty pSuper vector solution without an insert was added to the culture medium according to the protocol of FuGENE (registered trademark) HD transfection reagent (Promega E2311), and cultured at 37°C, 5% _CO2 for 24 hours.

Then, for both groups, 1 μg of a solution of pBI-CMV1MCS1 expressing "Firefly_2+2" under the control of the CMV promoter was added to the culture medium according to the protocol of FuGENE (registered trademark) HD transfection reagent (Promega E2311), cultured at 5% _CO2 and 37°C, and the cells were sampled at 8, 12, and 24 hours.

Regarding the protein expression of firefly luciferase, the luciferase luminescence intensity when "Firefly_2+2" was expressed for 24 hours in the group without adding Staple nucleic acid was set as the protein expression level 100%. The luciferase luminescence intensity of each sample was measured in comparison with this value, and the protein expression level ratio (%) was calculated.

The results are shown in Fig. 8-2. As also shown in the figure, the guanine quartet structure formed in the 3'-terminal UTR region due to the presence of the staple nucleic acid increased the protein expression level in the cell.

Example 7: Study on the target nucleic acid stabilization effect of the second generation Staple nucleic acid of the present invention

In this example, the second generation Staple nucleic acid was used to confirm whether the target nucleic acid stabilization effect was also produced when cells were used.

(7-1) Stabilization of mRNA

As described above, it was shown that the second-generation Staple nucleic acid of the present invention can play a role in stabilizing target nucleic acid in a cell-free system.

In this example, for neurofibromin 1 (NF1), a target nucleic acid having the following sequence "a portion of NF1 3'UTR" (target nucleic acid having the gene sequence of NF1) (SEQ ID NO: 46) was designed and used.

【Chemistry 27】

In this embodiment, the second generation of Staple nucleic acid acting on the target nucleic acid was prepared as follows. Its structure was designed according to the following method: using two guanine repeat sequences present in the 3'UTR of the target nucleic acid, two guanine repeat sequences were supplied by the Staple nucleic acid, and a guanine quartet structure was formed in the region corresponding to the 3'UTR of the target nucleic acid. It should be noted that the sequence of the Staple nucleic acid was transcribed by RNA pol III using the U6 series promoter in the cell. At this time, RNA polymerase will end transcription due to the continuous sequence of T, so part of T is replaced by A for use in the cell.

【Chemistry 28】

The method for investigating the stabilization of mRNA was basically investigated according to the method described in Example 6 (6-1). The prepared template RNA was used as the target nucleic acid, and the template RNA and the Staple nucleic acid were incubated at 90°C for 2 minutes in a composition of KCl (150mM) and Tris-HCl buffer (pH7.6) (10mM), and annealed by decreasing the temperature to 20°C at 1°C/min (template RNA: Staple = 1: 1.25). Using the annealing solution, a measurement solution (total volume: 150μl) was prepared in the form of RNA (0.04μM), 1×RNaseR buffer, RNase R (0.1 unit) or milliQ (control). Using a UV/VIS Spectrophotometer V-560 (JASCO), the absorption spectrum at 260nm was measured at 37°C for 1 hour. This was repeated 3 times and N=3. In this step, the UV absorption value increases when the target nucleic acid is decomposed, thereby enabling the detection of the decomposition of the target nucleic acid.

The results are shown in Figure 9-1 (the left figure of Figure 9-1 shows the second generation Staple nucleic acid, and the right figure of Figure 9-1 shows the results of the first generation Staple nucleic acid). By introducing the first generation Staple nucleic acid or the second generation Staple nucleic acid, it is possible to induce the formation of a guanine quadruple chain structure at the 3' end side "NF13'UTR" of the target nucleic acid, and the resistance of the target nucleic acid to 3'→5' exonuclease activity in the presence of RNaseR is also improved, and it can be confirmed that the stability of the target nucleic acid is improved (Figure 9-1). Among them, in the case of the combination of the target nucleic acid and the Staple nucleic acid, the second generation Staple nucleic acid further improves the stability of the target nucleic acid compared to the first generation Staple nucleic acid.

(7-2) Effect of increasing protein expression

In this example, it was confirmed whether the staple nucleic acid of the present invention can also form a guanine quartet structure in the target nucleic acid in cells and whether as a result, the stability of the target nucleic acid can be improved (and thus the protein expression can be increased) in cells.

In this example, the NF1 sequence used in (7-1) above was used as a target nucleic acid, and the second-generation staple nucleic acid was used as a staple nucleic acid.

MCF-7#9 cells were seeded at 1.0×10 ⁵ in a 10 cm culture dish and cultured for 72 hours in 10 ml of E-MEM medium containing 10% FBS and 1% penicillin. When 80% confluence was confirmed, the medium was replaced with 10 ml of E-MEM medium containing 10% FBS and cultured for 3 hours.

The cells were divided into two groups. The group using the Staple oligonucleotide was treated with pIRES expressing the sequence of the Staple oligonucleotide, and the group not using the Staple oligonucleotide was treated with 17 μg of an empty pIRES vector solution without an insert added to the culture medium according to the protocol of FuGENE (registered trademark) HD transfection reagent (Promega E2311). The cells were then cultured at 37°C at 5% _CO2 for 48 hours.

After culturing each cell for 48 hours, the medium was removed and the cells were washed twice with 5 ml of D-PBS (-). 350 μl of a mixed solution of RIPA buffer (Nacalai Tesque): protease inhibitor cocktail (Promega) = 99:1 was added to the culture dish, and the cells were recovered using a cell scraper. The cell lysate was passed through a 25G needle 10 times and centrifuged at 4°C for 10 minutes. The supernatant was transferred to a new tube, mixed with 6×SDS sample buffer (Nacalai Tesque), and heated at 95°C for 5 minutes.

The samples were separated by electrophoresis using Mini-PROTEAN TGX Gels (BIO-RAD) at 125 V and 126 mA for 110 minutes, and then blotted.

To the protein-printed membrane, a primary antibody against NF1 (neurofibromin 1D7R7D rabbit mAb #14623: Cell Signaling Technology) and a primary antibody against vinculin as a control (vinculin recombinant rabbit monoclonal antibody 42H89L44: Invitrogen) diluted to 1/1000 using Signal Enhancer HIKARI for Western Blotting and ELISA (Nacalai Tesque) were added and shaken at room temperature for 90 minutes.

After washing the membrane with PBS, a secondary antibody against rabbit IgG (#7074 anti-rabbit IgG, HRP-conjugated antibody: Cell Signaling Technology) diluted 1/1000 using Signal Enhancer HIKARI for Western Blotting and ELISA was added and shaken at room temperature for 60 minutes.

After washing the membrane with PBS, Chemi-Lumi One Super (Nacalai Tesque) was added to the membrane, and the membrane was left to stand at room temperature for 1 minute before detecting the bands with ImageQuant LAS 500 (GE Healthcare).

The results are shown in Fig. 9-2. When the staple nucleic acid was used, the protein expression of NF1, which is the target nucleic acid, increased significantly compared to when the staple nucleic acid was not used.

Example 8: Study on the ribosome diversion effect of the staple nucleic acid of the present invention in a single target nucleic acid (2)

In this example, the first-generation Staple nucleic acid or the second-generation Staple nucleic acid was used to confirm the occurrence of the ribosome diversion effect on a partial sequence of a single target nucleic acid.

In this example, a part of the sequence of the known firefly luciferase (F Luc) gene was changed to design a nucleotide sequence of a modified F Luc gene that serves as a template for mRNA (SEQ ID NO: 49) of a target nucleic acid of the first generation Staple nucleic acid. The modified F Luc gene mRNA serving as the target nucleic acid is designed so that when the first generation Staple nucleic acid of the present invention is hybridized and a guanine quartet structure is formed between the target nucleic acid and the Staple nucleic acid, a functional F Luc protein can be produced only when ribosome shunting occurs within the molecule of the target nucleic acid.

【Chemistry 29

In this embodiment, the first generation of Staple nucleic acids acting on the target nucleic acid was prepared as follows. Specifically, for these first generation Staple nucleic acids, two types of Staple nucleic acids (Staple nucleic acid B3 (sequence number 50), Staple nucleic acid B4 (sequence number 51)) were prepared, including the first nucleotide sequence and the second nucleotide sequence that hybridize to the Staple nucleic acid binding region shown in the underlined portion of the nucleotide sequence of the target nucleic acid.

【化３0】

In this example, a nucleotide sequence of a modified F Luc gene that serves as a template for mRNA (SEQ ID NO: 52) of a target nucleic acid as a second-generation Staple nucleic acid was also designed. The modified F Luc gene mRNA as the target nucleic acid was designed so that when the second-generation Staple nucleic acid of the present invention is hybridized and a guanine quartet structure is formed between the target nucleic acid and the Staple nucleic acid, a functional F Luc protein can be produced only when ribosome shunting occurs within the molecule of the target nucleic acid.

【Chemistry 31

In this embodiment, in addition, a second generation of Staple nucleic acid acting on the target nucleic acid was prepared as follows. Specifically, for these second generation Staple nucleic acids, a Staple nucleic acid (Staple nucleic acid B5 (SEQ ID NO. 53)) containing a first nucleotide sequence and a second nucleotide sequence hybridized in the Staple nucleic acid binding region shown in the underlined portion of the nucleotide sequence of the target nucleic acid and two guanine repeat sequences was prepared.

【化３2】

Using the modified F Luc gene mRNA (SEQ ID NO: 49 or SEQ ID NO: 52) as a template (1 μg), 20 μL of a cell-free protein expression mixture (RTS 100 Wheat Germ CECF Kit, 5 Prime, Inc) containing 0.14 μM of each Staple nucleic acid or not added was reacted at 24°C for 150 minutes. The obtained RNA was used as an mRNA template (1 μg), and 20 μL of a cell-free protein expression mixture (RTS100 Wheat Germ CECF Kit, 5 Prime, Inc) containing 0.14 μM of Staple nucleic acid or not was reacted at 24°C for 150 minutes. Fluorescence was measured using a fluorescence spectrophotometer (Jasco FP-8500) at an excitation wavelength of 480 nm and 25°C. As a positive control, a sample expressing F Luc based on the nucleotide sequence of the F Luc gene was used, and as a negative control, a sample expressing modified F Luc without applying Staple nucleic acid was used.

The results are shown in Fig. 10. In this figure, the FLuc signal intensity when FLuc is expressed based on the nucleotide sequence of the FLuc gene as a positive control is set to 100, and the FLuc signal intensity of the modified FLuc expression sample (without Staple nucleic acid), the modified FLuc expression sample in the presence of the first generation Staple nucleic acid (Staple nucleic acid B3 and Staple nucleic acid B4) (left figure), and the modified FLuc expression sample in the presence of the second generation Staple nucleic acid (Staple nucleic acid B5) (right figure) are compared. As a result, the FLuc signal intensity of the modified FLuc expression sample (without Staple nucleic acid) was 9.1 or 6.2% of the positive control, while the FLuc signal intensity when the first generation Staple nucleic acid was used was 68.4% (Staple nucleic acid B3) and 35.4% (Staple nucleic acid B4), and the FLuc signal intensity when the second generation Staple nucleic acid was used was 88.6% (Staple nucleic acid B5), and the intensity was increased.

Example 9: Study on the ribosome diversion effect between two target nucleic acids of the Staple nucleic acid of the present invention

In this example, the first-generation Staple nucleic acid and the second-generation Staple nucleic acid were used to confirm the occurrence of the ribosome diversion effect at the partial sequence between two target nucleic acids.

Taking the first generation of Staple nucleic acid as an example, the outline of the method for confirming ribosome diversion between two target nucleic acids is shown in the following scheme. Specifically, a guanine quartet structure is formed between the fragment of gene A and the fragment of gene B through the Staple nucleic acid as in (1), and the confirmation is performed by investigating whether the ribosome crosses from the RNA gene of gene A to the RNA of B at the position of the guanine quartet structure. A nucleotide sequence that is functional through the combination of gene A and gene B if ribosome diversion occurs at the corresponding position is prepared, thereby confirming that the RNA of gene A crosses to the RNA of gene B. It should be noted that (2) to (4) all serve as controls that cannot form a guanine quartet structure.

【化33】

In order to study whether the first-generation Staple nucleic acid causes ribosome shunting, the following gene A and gene B sequences were designed as target sequences.

【Chemistry 34

【化３5】

In this example, first-generation staple nucleic acids used in the method (1) and control nucleic acids used in the method (2) for these target nucleic acids were prepared as follows.

【化３6】

In order to study whether the second-generation Staple nucleic acid causes ribosome shunting, the following gene A and gene B sequences were designed as target sequences.

【化37】

【化38】

In this example, second-generation staple nucleic acids (staple nucleic acids C3) for use in methods (1) and (4) and control nucleic acids (control nucleic acids C4) for use in method (2) for these target nucleic acids were prepared as follows.

【化３9】

The above-mentioned gene A and the above-mentioned gene B were mixed at a concentration ratio of 1:1, 1.5 equivalents of Staple nucleic acid were added thereto, and the temperature was lowered by 1°C per minute from 90°C for annealing. Here, regarding the first-generation Staple nucleic acid, Staple nucleic acid C1 was used under the condition of (1), Staple nucleic acid C2 was used under the condition of (2), no Staple nucleic acid was added under the condition of (3), and Staple nucleic acid C1 was used under the condition of (4).

For the total amount of 1 μg of the complex mRNA, 12.5 μl of mRNA (1 μg) was used in a cell-free protein synthesis reaction solution (RTS100 Wheat Germ Kit, biotechrabbit). After incubating the reaction solution at 24°C for 1 hour, the luciferase activity was evaluated using a luciferase assay kit (Promega) and a POWERSCAN·H1 microplate reader (BioTek).

The results are shown in Figure 11. As shown in the figure, in both the first-generation Staple nucleic acid and the second-generation Staple nucleic acid, the fluorescence of luciferase was almost undetectable in the cases of (2) to (4), while the fluorescence of luciferase was strongly detected in the case of (1). This result indicates that when the Staple nucleic acid of the present invention is used to form a guanine quadruple chain structure between two molecules, the ribosome will be diverted between the two molecules at this portion.

Industrial Applicability

The present invention can develop and provide a new Staple nucleic acid (second-generation Staple nucleic acid), which supplies guanine repeat sequences through oligonucleotides, resulting in the formation of a guanine quartet structure on the target nucleic acid using a total of four guanine repeat sequences, namely, guanine repeats on the target nucleic acid and guanine repeat sequences on the oligonucleotide. The present invention can also provide new uses of Staple nucleic acids for first-generation Staple nucleic acids and second-generation Staple nucleic acids.

Claims (25)

1. A G-supplied oligonucleotide comprising a 1 st nucleotide sequence and a 2 nd nucleotide sequence hybridized with nucleotide sequences on the 5 'side or 3' side of a guanine repetition sequence for a nucleotide sequence portion comprising 1 st to 3 rd guanine repetition sequences on a target nucleic acid, and 3 rd to 1 st guanine repetition sequences depending on the number of guanine repetition sequences on the target nucleic acid,

The G-supply oligonucleotide is capable of changing the three-dimensional structure of a target nucleic acid by hybridization of the 1 st nucleotide sequence and the 2 nd nucleotide sequence with the target nucleic acid, shortening the spatial distance between the guanine repetitive sequence on the target nucleic acid and the guanine repetitive sequence on the G-supply oligonucleotide at the total 4 positions, and forming a guanine quadruplex structure by the guanine repetitive sequences at the 4 positions.

2. The G-supply oligonucleotide according to claim 1, wherein the 1 st nucleotide sequence and the 2 nd nucleotide sequence of the G-supply oligonucleotide are identical to the 5' -side or 3' -side nucleotide sequence portion of the guanine repeating sequence on the 3' -terminal side of the guanine repeating sequence on the target nucleic acid,

Nucleotide sequence portion on 5' -side or 3' -side of guanine repetitive sequence on 5' -terminal side

Hybridization is performed.

3. The G-supplied oligonucleotide of claim 1 or 2, which is DNA, RNA, a modified nucleic acid, or a combination of these.

4. A pharmaceutical composition comprising a G-donating oligonucleotide comprising a1 st nucleotide sequence and a2 nd nucleotide sequence hybridized with a nucleotide sequence on the 5 'side or 3' side of a guanine repetitive sequence for a nucleotide sequence portion comprising 1 st to 3 rd guanine repetitive sequences on a target nucleic acid, and 3 rd to 1 st guanine repetitive sequences depending on the number of guanine repetitive sequences on the target nucleic acid,

The G-supply oligonucleotide is capable of changing the three-dimensional structure of a target nucleic acid by hybridization of the 1 st nucleotide sequence and the 2 nd nucleotide sequence with the target nucleic acid, shortening the spatial distance between the guanine repetitive sequence on the target nucleic acid and the guanine repetitive sequence on the G-supply oligonucleotide at the total 4 positions, and forming a guanine quadruplex structure by the guanine repetitive sequences at the 4 positions.

5. The pharmaceutical composition according to claim 4, wherein the 1 st nucleotide sequence and the 2 nd nucleotide sequence of the G-donating oligonucleotide are identical to the 5' -side or 3' -side nucleotide sequence portion of the guanine repeating sequence at the 3' -terminal side of the guanine repeating sequence on the target nucleic acid,

Nucleotide sequence portion on 5' -side or 3' -side of guanine repetitive sequence on 5' -terminal side

Hybridization is performed.

6. The pharmaceutical composition of claim 4 or 5, wherein is DNA, RNA, a modified nucleic acid, or a combination of these.

7. A method for producing a G-supplied oligonucleotide comprising a1 st nucleotide sequence and a2 nd nucleotide sequence hybridized with a nucleotide sequence on the 5 'side or 3' side of a guanine repetition sequence with respect to a nucleotide sequence portion comprising 1 st to 3 rd guanine repetition sequences on a target nucleic acid, and 3 rd to 1 st guanine repetition sequences depending on the number of guanine repetition sequences on the target nucleic acid,

The method is designed such that a G-supply oligonucleotide is designed and synthesized such that the G-supply oligonucleotide can be folded such that the 1 st nucleotide sequence and the 2 nd nucleotide sequence of the G-supply oligonucleotide hybridize with a target nucleic acid, thereby shortening the distance between the guanine repeats on the target nucleic acid and the space between the guanine repeats at 4 positions in total on the G-supply oligonucleotide.

8. The method for producing a G-supply oligonucleotide according to claim 7, wherein the 1 st nucleotide sequence and the 2 nd nucleotide sequence of the G-supply oligonucleotide are identical to a nucleotide sequence portion on the 5' side or 3' side of a guanine repeating sequence on the.3 ' -terminal side of the guanine repeating sequence on the target nucleic acid,

Nucleotide sequence portion on 5' -side or 3' -side of guanine repetitive sequence on 5' -terminal side

Hybridization is performed.

9. The method for producing a G-supplied oligonucleotide according to claim 7 or 8, wherein it is DNA, RNA, a modified nucleic acid, or a combination of these.

10. A method for inhibiting the expression of a protein by a target nucleic acid, wherein a G-donating oligonucleotide changes the steric structure of the target nucleic acid by hybridizing a1 st nucleotide sequence and a 2 nd nucleotide sequence with the target nucleic acid, shortens the spatial distance of guanine repeats at 4 positions in total on the target nucleic acid and on the G-donating oligonucleotide, and the guanine repeats at 4 positions form a guanine quadruplex structure, thereby inhibiting the expression of the protein by the target nucleic acid,

The G-supply oligonucleotide comprises a 1 st nucleotide sequence and a 2 nd nucleotide sequence hybridized with nucleotide sequences on the 5 'side or the 3' side of a guanine repetitive sequence aiming at a nucleotide sequence part containing 1-3 guanine repetitive sequences on a target nucleic acid, and 3-1 guanine repetitive sequences which depend on the number of the guanine repetitive sequences on the target nucleic acid.

11. The inhibition method according to claim 10, wherein the 1 st nucleotide sequence and the 2 nd nucleotide sequence of the G-donating oligonucleotide are mixed with a nucleotide sequence portion on the 5' side or 3' side of the guanine repeating sequence on the 3' -terminal side of the guanine repeating sequence on the target nucleic acid,

Nucleotide sequence portion on 5' -side or 3' -side of guanine repetitive sequence on 5' -terminal side

Hybridization is performed.

12. The inhibition method according to claim 10 or 11, wherein it is DNA, RNA, a modified nucleic acid or a combination of these.

13. The inhibition method according to claim 10 or 11, wherein the inhibition of protein expression is based on inhibition of a reverse transcription reaction or inhibition of a protein translation reaction.

14. A protein expression inhibition kit comprising a G-donating oligonucleotide,

The G-supply oligonucleotide comprises a 1 st nucleotide sequence and a 2 nd nucleotide sequence hybridized with nucleotide sequences on the 5 'side or the 3' side of a guanine repetitive sequence for a nucleotide sequence part comprising 1 st to 3 rd guanine repetitive sequences on a target nucleic acid, and 3 rd to 1 st guanine repetitive sequences depending on the number of guanine repetitive sequences on the target nucleic acid,

The G-supply oligonucleotide can change the three-dimensional structure of the target nucleic acid by hybridizing the 1 st nucleotide sequence and the 2 nd nucleotide sequence with the target nucleic acid, so that the space distance between the guanine repetitive sequence on the target nucleic acid and the guanine repetitive sequence on the G-supply oligonucleotide is shortened at the total 4 positions of the guanine repetitive sequences, and the guanine repetitive sequences at the 4 positions form a guanine four-chain structure, thereby inhibiting the expression of proteins by the target nucleic acid.

15. The protein expression suppression kit according to claim 14, wherein the 1 st nucleotide sequence and the 2 nd nucleotide sequence of the G-supply oligonucleotide are linked to a nucleotide sequence portion on the 5' side or 3' side of the guanine repetitive sequence on the 3' -terminal side of the guanine repetitive sequence on the target nucleic acid,

Nucleotide sequence portion on 5' -side or 3' -side of guanine repetitive sequence on 5' -terminal side

Hybridization is performed.

16. The protein expression inhibition kit according to claim 14 or 15, wherein the inhibition of protein expression is based on inhibition of a reverse transcription reaction or inhibition of a protein translation reaction.

17. A method for producing an altered protein from a target nucleic acid, wherein an oligonucleotide alters the steric structure of the target nucleic acid by hybridizing a1 st nucleotide sequence and a2 nd nucleotide sequence to the target nucleic acid, shortens the spatial distance of guanine repeats on the target nucleic acid and guanine repeats on the oligonucleotide at a total of 4, the guanine repeats at 4 forming a guanine quadruple structure, thereby forming a loop portion of the target nucleic acid, branching ribosomes to the loop portion, and producing the altered protein from the target nucleic acid,

The oligonucleotide comprises a1 st nucleotide sequence and a2 nd nucleotide sequence hybridized with nucleotide sequences on the 5 'side or the 3' side of a guanine repetitive sequence aiming at a nucleotide sequence part containing 1-4 guanine repetitive sequences on target nucleic acid, and 3-0 guanine repetitive sequences depending on the number of the guanine repetitive sequences on the target nucleic acid.

18. The method for producing an altered protein according to claim 17, wherein the 1 st nucleotide sequence and the 2 nd nucleotide sequence of the oligonucleotide are identical to the 5' side or 3' side nucleotide sequence portion of the guanine repeating sequence on the 3' terminal side of the guanine repeating sequence on the target nucleic acid,

Nucleotide sequence portion on 5' -side or 3' -side of guanine repetitive sequence on 5' -terminal side

Hybridization is performed.

19. A method of making an engineered protein according to claim 17 or 18, wherein is DNA, RNA, a modified nucleic acid or a combination of these.

20. A method for producing a fusion protein, wherein the 1 st nucleotide sequence in an oligonucleotide hybridizes to the 1 st target nucleic acid, the 2 nd nucleotide sequence hybridizes to the 2 nd target nucleic acid and the oligonucleotide alters the steric structure of these target nucleic acids such that the spatial distance of the guanine repetitive sequence at 1 to 2 positions on the 1 st target nucleic acid, the guanine repetitive sequence at 1 to 2 positions on the 2 nd target nucleic acid and the guanine repetitive sequence at 2 to 0 positions on the oligonucleotide add up to 4 guanine repetitive sequences is shortened, the 4 guanine repetitive sequences form a guanine quadruple structure, thereby shunting ribosomes between the 1 st target nucleic acid and the 2 nd target nucleic acid, thereby producing a fusion protein from a translation product of a partial sequence of the 1 st target nucleic acid and a translation product of a partial sequence of the 2 nd target nucleic acid,

The oligonucleotide comprises:

A 1 st nucleotide sequence hybridized with a nucleotide sequence on the 5 'side or 3' side of a guanine repetitive sequence, which is a nucleotide sequence portion comprising 1 to 2 guanine repetitive sequences on the 1 st target nucleic acid,

For a nucleotide sequence portion comprising a guanine repetition sequence of 1 to 2 on a2 nd target nucleic acid, a2 nd nucleotide sequence hybridized with a nucleotide sequence of 5 'side or 3' side of the guanine repetition sequence, and 2 to 0 guanine repetition sequences depending on the number of guanine repetition sequences on the 1 st target nucleic acid and the number of guanine repetition sequences on the 2 nd target nucleic acid.

21. The method for producing a fusion protein according to claim 20, wherein the 1 st nucleotide sequence and the 2 nd nucleotide sequence of the oligonucleotide are linked to a nucleotide sequence portion on the 5' side or 3' side of the guanine repeating sequence on the 3' -terminal side of the guanine repeating sequence on the target nucleic acid,

Nucleotide sequence portion on 5' -side or 3' -side of guanine repetitive sequence on 5' -terminal side

Hybridization is performed.

22. A method of making a fusion protein according to claim 20 or 21, wherein is DNA, RNA, a modified nucleic acid or a combination of these.

23. A method for stabilizing a target nucleic acid, wherein an oligonucleotide alters the steric structure of the target nucleic acid by hybridizing a1 st nucleotide sequence and a2 nd nucleotide sequence to the target nucleic acid, shortens the spatial distance of guanine repeats at 4 positions in total of the guanine repeats on the target nucleic acid and the guanine repeats on the oligonucleotide, and the guanine repeats at 4 positions form a guanine quadruple structure, thereby inhibiting binding of a nucleic acid hydrolase to the target nucleic acid or inhibiting the function of a nucleic acid hydrolase, thereby stabilizing the target nucleic acid,

The oligonucleotide comprises a1 st nucleotide sequence and a2 nd nucleotide sequence hybridized with nucleotide sequences on the 5 'side or the 3' side of a guanine repetitive sequence aiming at a nucleotide sequence part containing 1-4 guanine repetitive sequences on target nucleic acid, and 3-0 guanine repetitive sequences depending on the number of the guanine repetitive sequences on the target nucleic acid.

24. The method for stabilizing a target nucleic acid according to claim 23, wherein the 1 st nucleotide sequence and the 2 nd nucleotide sequence of the oligonucleotide are identical to a nucleotide sequence portion on the 5' side or 3' side of a guanine repeating sequence on the 3' terminal side of the guanine repeating sequence on the target nucleic acid,

Nucleotide sequence portion on 5' -side or 3' -side of guanine repetitive sequence on 5' -terminal side

Hybridization is performed.

25. The method of stabilizing a target nucleic acid of claim 23 or 24, wherein is DNA, RNA, a modified nucleic acid, or a combination of these.